RESUMEN
The global COVID-19 pandemic ignited an unprecedented race to develop vaccines and antibody therapeutics. AstraZeneca's pursuit to provide AZD7442 (EVUSHELD), two long-acting, SARS-CoV-2 spike receptor binding domain-specific neutralizing monoclonal antibodies, to individuals at risk on highly accelerated timelines challenged our traditional ways of process development and spurred the rapid adoption of novel approaches. Conventional upstream development processes were replaced by agile strategies that combined technological advances and highly accelerated workflows. With calculated business risks and close cross-functional collaborations, this process paved the way for hyper accelerated antibody development from discovery through manufacturing, process validation, emergency use authorization filing, and global regulatory approvals. The result was initiation of commercial manufacturing at a contract manufacturing organization less than 6 months from the selection of cilgavimab and tixagevimab-a process that historically has taken close to 10 years.
RESUMEN
MOTIVATION: Recombinant DNA technology is widely used for different applications in biology, medicine and bio-technology. Viral transduction and plasmid transfection are among the most frequently used techniques to generate recombinant cell lines. Many of these methods result in the random integration of the plasmid into the host genome. Rapid identification of the integration sites is highly desirable in order to characterize these engineered cell lines. RESULTS: We developed detectIS: a pipeline specifically designed to identify genomic integration sites of exogenous DNA, either a plasmid containing one or more transgenes or a virus. The pipeline is based on a Nextflow workflow combined with a Singularity image containing all the necessary software, ensuring high reproducibility and scalability of the analysis. We tested it on simulated datasets and RNA-seq data from a human sample infected with Hepatitis B virus. Comparisons with other state of the art tools show that our method can identify the integration site in different recombinant cell lines, with accurate results, lower computational demand and shorter execution times. AVAILABILITY AND IMPLEMENTATION: The Nextflow workflow, the Singularity image and a test dataset are available at https://github.com/AstraZeneca/detectIS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
ARN , Programas Informáticos , Humanos , Reproducibilidad de los Resultados , Genómica , ADNRESUMEN
Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.
Asunto(s)
Vías Secretoras , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Proteínas Recombinantes , Vías Secretoras/genéticaRESUMEN
Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed 'regulation elements (RgE)', in the 5'-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5'-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5'-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.
Asunto(s)
Regiones no Traducidas 5' , Regulación de la Expresión Génica/genética , Biosíntesis de Proteínas/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Animales , Células CHO , Cerebrósido Sulfatasa/genética , Cerebrósido Sulfatasa/metabolismo , Cricetulus , Expresión Génica , Vectores Genéticos , Células HEK293 , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Secuencias Invertidas Repetidas , Conformación de Ácido Nucleico , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Proteínas Recombinantes/biosíntesisRESUMEN
Host cell proteins (HCP) that co-purify with biologics produced in Chinese hamster ovary cells have been shown to impact product quality through proteolytic degradation of recombinant proteins, leading to potential product losses. Several problematic HCPs can remain in the final product even after extensive purification. Each recombinant cell line has a unique HCP profile that can be determined by numerous upstream and downstream factors, including clonal variation and the protein sequence of the expressed therapeutic molecule. Here, we worked with recombinant cell lines with high levels of copurifying HCPs, and showed that in those cell lines even modest downregulation (≤50%) of the difficult to remove HCP Cathepsin D, through stable short hairpin RNA interference or monoallelic deletion of the target gene using CRISPR-Cas9, is sufficient to greatly reduce levels of co-purifying HCP as measured by high throughput targeted LC-MS. This reduction led to improved product quality by reducing fragmentation of the drug product in forced degradation studies to negligible levels. We also show the potential of cell engineering to target other undesired HCPs and relieve the burden on downstream purification.
Asunto(s)
Anticuerpos Monoclonales , Sistemas CRISPR-Cas , Expresión Génica , Ingeniería Metabólica , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Células CHO , Cricetulus , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. With this in mind, we generated a novel hydrogen peroxide evolved host using directed host cell evolution. Here we demonstrate that this host has heritable resistance to hydrogen peroxide over many generations, displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defense genes such as those involved in glutathione synthesis and turnover, and has improved glutathione content. Additionally, we show that this host has significantly improved transfection recovery times, improved growth and viability properties in a fed-batch production process, and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells. These findings demonstrate that host cell evolution represents a powerful methodology for improving specific host cell characteristics that can positively impact the expression of difficult to express biotherapeutics.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Células CHO , Peróxido de Hidrógeno , Animales , Células CHO/clasificación , Cricetulus , Estrés Oxidativo , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass. In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts.
Asunto(s)
Células CHO/metabolismo , Ingeniería Metabólica/métodos , Proteínas Recombinantes/biosíntesis , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Animales , Cricetulus , Proteínas Recombinantes/genéticaRESUMEN
Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation.
Asunto(s)
Simulación por Computador , Regiones Promotoras Genéticas , Animales , Sitios de Unión , Células CHO , Cricetulus , Biología Sintética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes: Energy generation, redox control, and synthesis of cell and product biomass. However, very little is known about the activity of different functional classes of AATs in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro. In this study, we have comparatively characterized AAT expression in untransfected and monoclonal antibody (MAb)-producing CHO cells using transcriptome analysis by RNA-seq, and mechanistically dissected AAT function using a variety of transporter-specific chemical inhibitors, comparing their effect on cell proliferation, recombinant protein production, and amino acid transport. Of a possible 56 mammalian plasma membrane AATs, 16 AAT messenger RNAs (mRNAs) were relatively abundant across all CHO cell populations. Of these, a subset of nine AAT mRNAs were more abundant in CHO cells engineered to produce a recombinant MAb. Together, upregulated AATs provide additional supply of specific amino acids overrepresented in MAb biomass compared to CHO host cell biomass, enable transport of synthetic substrates for glutathione synthesis, facilitate transport of essential amino acids to maintain active protein synthesis, and provide amino acid substrates for coordinated antiport systems to maintain supplies of proteinogenic and essential amino acids.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/metabolismo , Animales , Reactores Biológicos , Células CHO , Cricetinae , Cricetulus , Medios de Cultivo/química , Glutamato-Amoníaco Ligasa/metabolismoRESUMEN
Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.
Asunto(s)
Empalme Alternativo , Inmunoglobulina G , Animales , Cricetinae , Intrones/genética , Cricetulus , Células CHO , Inmunoglobulina G/genéticaRESUMEN
Cell line development is an essential stage in biopharmaceutical development that often lies on the critical path. Failure to fully characterise the lead clone during initial screening can lead to lengthy project delays during scale-up, which can potentially compromise commercial manufacturing success. In this study, we propose a novel cell line development methodology, referenced as CLD 4, which involves four steps enabling autonomous data-driven selection of the lead clone. The first step involves the digitalisation of the process and storage of all available information within a structured data lake. The second step calculates a new metric referenced as the cell line manufacturability index (MI CL) quantifying the performance of each clone by considering the selection criteria relevant to productivity, growth and product quality. The third step implements machine learning (ML) to identify any potential risks associated with process operation and relevant critical quality attributes (CQAs). The final step of CLD 4 takes into account the available metadata and summaries all relevant statistics generated in steps 1-3 in an automated report utilising a natural language generation (NLG) algorithm. The CLD 4 methodology was implemented to select the lead clone of a recombinant Chinese hamster ovary (CHO) cell line producing high levels of an antibody-peptide fusion with a known product quality issue related to end-point trisulfide bond (TSB) concentration. CLD 4 identified sub-optimal process conditions leading to increased levels of trisulfide bond that would not be identified through conventional cell line development methodologies. CLD 4 embodies the core principles of Industry 4.0 and demonstrates the benefits of increased digitalisation, data lake integration, predictive analytics and autonomous report generation to enable more informed decision making.
RESUMEN
When expressing complex biotherapeutic proteins, traditional expression plasmids and methods may not always yield sufficient levels of high-quality product. High-strength viral promoters commonly used for recombinant protein (rProtein) production in mammalian cells allow for maximal expression, but provide limited scope to alter their transcription dynamics. However, synthetic promoters designed to provide tunable transcriptional activity offer a plasmid engineering approach to more precisely regulate product quality, yield or to reduce product related contaminants. We substituted the viral promoter CMV with synthetic promoters that offer different transcriptional activities to express our gene of interest in Chinese hamster ovary (CHO) cells. Stable pools were established and the benefits of regulating transgene transcription on the quality of biotherapeutics were examined in stable pool fed-batch overgrow experiments. Specific control of gene expression of the heavy chain (HC):light chain (LC) of a Fab, and the ratio between the two HCs in a Duet mAb reduced levels of aberrant protein contaminants; and the controlled expression of the helper gene XBP-1s improved expression of a difficult-to-express mAb. This synthetic promoter technology benefits applications that require custom activity. Our work highlights the advantages of employing synthetic promoters for production of more complex rProteins.
RESUMEN
Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post-translational modifications. A potential issue when utilizing CHO cells for therapeutic protein production is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS-CHO cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this cell line, stability of expression was retained for 40-50 generations after which time a 40% loss in antibody production was detected. The instability observed within the cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative cell time values and an apparent increased sensitivity to cellular stress (exemplified by increased mRNA expression of the stress-inducible gene GADD153). Changes were also noted in cellular metabolism during LTC (alterations to extracellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO cells during LTC ranging from instability of recombinant target production at a post-mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning cellular phenotype changes require further detailed analysis at a molecular level.
Asunto(s)
Anticuerpos/metabolismo , Biotecnología/métodos , Animales , Anticuerpos/genética , Células CHO , Cricetinae , Cricetulus , Inestabilidad Genómica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
During early stage development of a therapeutic IgG1 monoclonal antibody, high levels of low molecular weight (LMW) peaks were observed by high performance size-exclusion chromatography and capillary electrophoresis. Further characterization of the LMW peak enriched HPSEC fractions using reversed phase liquid chromatography coupled to mass spectrometry showed these LMW species were 47 kDa and 50 kDa in size. However, the measured masses could not be matched to any fragments resulting from peptide bond hydrolysis. To identify these unknown LMW species, molecular characterization methods were employed, including high-throughput sequencing of RNA. Transcriptomic analysis revealed the LMW species were generated by mis-splicing events in the heavy chain transcript, which produced truncated heavy chain products that assembled with the light chain to mimic the appearance of fragments identified by routine purity assays. In an effort to improve product quality, an optimized purification process was developed. Characterization of the process intermediates confirmed removal of both LMW species by the optimized process. Our study demonstrates that deep-dive analytical characterization of biotherapeutics is critical to ensure product quality and inform process development. Transcriptomic analysis tools can help identify the cause of unknown species, and plays a key role in product and process characterization.
Asunto(s)
Anticuerpos Monoclonales , Cromatografía de Fase Inversa , Anticuerpos Monoclonales/química , Cromatografía de Fase Inversa/métodos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Péptidos , ARNRESUMEN
With the aim of increasing protein productivity of Chinese hamster ovary (CHO) cells, we sought to generate new CHO hosts with favorable biomanufacturing phenotypes and improved functionality. Here, we present an innovative approach of enriching the CHO host cells with a high mitochondrial membrane potential (MMP). Stable transfectant pools and clonal cell lines expressing difficult-to-express bispecific molecules generated from the MMP-enriched host outperformed the parental host by displaying (1) improved fed-batch productivity; (2) enhanced long-term cell viability of pools; (3) more favorable lactate metabolism; and (4) improved cell cloning efficiency during monoclonal cell line generation. Proteomic analysis together with Western blot validation were used to investigate the underlying mechanisms by which high MMP influenced production performance. The MMP-enriched host exhibited multifaceted protection against mitochondrial dysfunction and endoplasmic reticulum stress. Our findings indicate that the MMP-enriched host achieved an overall "fitter" phenotype that contributes to the significant improvement in biomanufacturing capability.
Asunto(s)
Potencial de la Membrana Mitocondrial/genética , Ingeniería Metabólica , Mitocondrias/genética , Mitocondrias/metabolismo , Animales , Células CHO , CricetulusRESUMEN
Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression- and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.
Asunto(s)
Anticuerpos Biespecíficos , Animales , Anticuerpos Biespecíficos/metabolismo , Autofagia , Células CHO , Cricetinae , Cricetulus , Biología de SistemasRESUMEN
Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.
Asunto(s)
Estrés del Retículo Endoplásmico , Eritropoyetina , Animales , Estrés del Retículo Endoplásmico/fisiología , Eritropoyetina/genética , Eritropoyetina/metabolismo , Células HEK293/metabolismo , Humanos , Mamíferos/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
This article describes how community-based participatory research (CBPR) led to the discovery of the unintended consequences of jail and prison copayment policy on women prisoners' health. The article addresses (a) a working definition of participatory research; (b) the importance of research with women prisoners; (c) the origins and development of our work and its grounding in CBPR; (d) issues related to research with prisoners; and (e) recommendations for using participatory methods to bring women prisoners into the discourse about the practices and policies that impact their lives. These methods have the potential to minimize the invisibility of prisoners and their health disparities.
Asunto(s)
Investigación Participativa Basada en la Comunidad , Prisioneros , Calidad de la Atención de Salud , Salud de la Mujer , Adulto , California , Femenino , Accesibilidad a los Servicios de Salud , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
To successfully engineer mammalian cells for a desired purpose, multiple recombinant genes are required to be coexpressed at a specific and optimal ratio. In this study, we hypothesized that synthetic promoters varying in transcriptional activity could be used to create single multigene expression vectors coexpressing recombinant genes at a predictable relative stoichiometry. A library of 27 multigene constructs was created comprising three discrete fluorescent reporter gene transcriptional units in fixed series, each under the control of either a relatively low, medium, or high transcriptional strength synthetic promoter in every possible combination. Expression of each reporter gene was determined by absolute quantitation qRT-PCR in CHO cells. The synthetic promoters did generally function as designed within a multigene vector context; however, significant divergences from predicted promoter-mediated transcriptional activity were observed. First, expression of all three genes within a multigene vector was repressed at varying levels relative to coexpression of identical reporter genes on separate single gene vectors at equivalent gene copies. Second, gene positional effects were evident across all constructs where expression of the reporter genes in positions 2 and 3 was generally reduced relative to position 1. Finally, after accounting for general repression, synthetic promoter transcriptional activity within a local multigene vector format deviated from that expected. Taken together, our data reveal that mammalian synthetic promoters can be employed in vectors to mediate expression of multiple genes at predictable relative stoichiometries. However, empirical validation of functional performance is a necessary prerequisite, as vector and promoter design features can significantly impact performance.
Asunto(s)
Ingeniería Celular/métodos , Expresión Génica , Familia de Multigenes , Regiones Promotoras Genéticas/genética , Activación Transcripcional , Animales , Células CHO , Cricetulus , Biblioteca de Genes , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/genética , Plásmidos/genética , Proteína Fluorescente RojaRESUMEN
Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.