Background
When HEK293T cells are used to produce recombinant proteins, endogenous proteins within the host cells are released and mixed into the final product during cell lysis and protein extraction, which can have an impact on the purity and safety of the product. We refer to such endogenous proteins as host cell proteins (HCPs), and HEK293T cells contain a wide variety of HCPs, including enzymes, structural proteins, signaling proteins, and other metabolism-related proteins, and thus also have a multifaceted impact on the final bioproduct. For example, the immunogenicity of HCPs triggers an immune response, which in the application of biologics such as therapeutic antibodies, may lead to the development of resistance or other immune side effects in patients. The presence of enzymes, such as proteases and phosphatases, in HCPs may degrade or modify the target proteins, affecting the stability and activity of the proteins, and consequently, affecting their function.
Therefore the detection and removal of HCPs is a critical step in the production process of biologics. In order to ensure the accuracy and reliability of the detection, a combination of techniques is often used to detect HCPS, the most commonly used methods being ELISA, mass spectrometry and WB. ELISA assay has high sensitivity and high throughput through the use of polyclonal antibodies to specifically recognize HCPs. Mass spectrometry provides a detailed profile of host cell proteins and helps to identify specific interfering components. On the other hand the removal of HCPs is also a major concern and is now commonly performed by multi-step purification processes, such as the application of multiple chromatographic techniques (e.g., ion-exchange chromatography, affinity chromatography, and hydrophobic chromatography) in order to achieve optimal purification. Protein engineering techniques can also be used to design target proteins that are easier to purify, e.g., by adding specific tags to the protein sequence to facilitate capture and purification.
Additionally researchers are influencing the growth and production properties of HEK293 cells by modifying the expression of specific genes, which in turn increases the production of target proteins and reduces the impact of HCPs. Specific genes in cellular pathways involved in protein biosynthesis can be temporarily or permanently modified in a targeted manner, for example, through the use of RNA interference, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN). Global genetic or strain engineering and genome-wide analysis tools, such as random mutagenesis and RNA interference screening, can also be employed to identify novel target molecules involved in protein biosynthesis.
Figure 1. Engineering the HEK293 cell line for improved culture performance
(Source: Abaandou L, et al. 2021)
Alternative Names
HEK293T host cell protein ELISA Kit
References
- 1. Abaandou L, et al. Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes. Cells. 2021 Jul 2;10(7):1667.
- 2. Zhang Y, et al. Development of stable HEK293T cell pools expressing CSFV E2 protein: A potential antigen expression platform. Vaccine. 2023 Feb 24;41(9):1573-1583.
References
HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors
Front Bioeng Biotechnol
Authors: Tan E, Chin CSH, Lim ZFS, Ng SK.
Abstract
Animal cell-based expression platforms enable the production of complex biomolecules such as recombinant proteins and viral vectors. Although most biotherapeutics are produced in animal cell lines, production in human cell lines is expanding. One important advantage of using human cell lines is the increased potential that the resulting biotherapeutics would carry more "human-like" post-translational modifications. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. In this review, we discuss the use of HEK293 cells and its subtypes in the production of biotherapeutics. We also compare their usage against other commonly used host cell lines in each category of biotherapeutics and summarise the factors influencing the choice of host cell lines used.
ROP18-Mediated Transcriptional Reprogramming of HEK293T Cell Reveals New Roles of ROP18 in the Interplay Between Toxoplasma gondii and the Host Cell
Front Cell Infect Microbiol
Authors: Li JX, He JJ, Elsheikha HM, Ma J, Xu XP, Zhu XQ.
Abstract
Toxoplasma gondii secretes a number of virulence-related effector proteins, such as the rhoptry protein 18 (ROP18). To further broaden our understanding of the molecular functions of ROP18, we examined the transcriptional response of human embryonic kidney cells (HEK293T) to ROP18 of type I T. gondii RH strain. Using RNA-sequencing, we compared the transcriptome of ROP18-expressing HEK293T cells to control HEK293T cells. Our analysis revealed that ROP18 altered the expression of 750 genes (467 upregulated genes and 283 downregulated genes) in HEK293T cells. Gene ontology (GO) and pathway enrichment analyses showed that differentially expressed genes (DEGs) were significantly enriched in extracellular matrix- and immune-related GO terms and pathways. KEGG pathway enrichment analysis revealed that DEGs were involved in several disease-related pathways, such as nervous system diseases and eye disease. ROP18 significantly increased the alternative splicing pattern "retained intron" and altered the expression of 144 transcription factors (TFs). These results provide new insight into how ROP18 may influence biological processes in the host cells via altering the expression of genes, TFs, and pathways. More in vitro and in vivo studies are required to substantiate these findings.
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