HEK293T HCP ELISA Kit

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)

HEK293T host cell protein ELISA Kit