E.coli cells are widely used for the production of biopharmaceuticals. During drug development, the analysis of residual Host Cell Proteins (HCPs) is important for ensuring the quality and safety of biopharmaceuticals. For E.coli, the HCP pattern is strongly influenced by the applied manufacturing conditions. A traditional generic assay with only one set of antibodies does not adequately represent this diversity. To overcome limitations connected to traditional generic HCP assays, BioGenes has developed the enhanced generic E.coli|360-HCP ELISA, which is composed of a set of four different generic kits.
1) Genetic background of E.coli strains
The genomes of different E.coli strains show remarkable diversity. The typical E.coli genome encodes for approx. 4,500 - 5,000 genes, from which approx. 50 % of the genes are represented in the core genome. It is estimated that the pan-genome of E.coli comprises approx. 128,000 genes, and thus carries a large number of potential HCP species.
2) Cell cultivation conditions
The gene expression of E.coli is highly dependent on the growth phase of the cells, type of media and supplements and growth conditions. The re-organization of central metabolic pathways, the expression of stress-related genes and the secretion of enzymes for substrate-degradation are mainly influenced by the availability of nutrients and the presence of cell stress conditions such as temperature, oxygen and salinity.
3) Properties of the recombinant drug
The over-expression of a recombinant (foreign) protein can lead to significant changes in the proteome of the recombinant strain with respect to the expression patterns of metabolically functional proteins, chaperones and heat shock proteins.
All these conditions together influence the unique HCP profile which occurs during E.coli fermentation, and no single generic kit will be able to detect every HCP.
The E.coli|360-HCP ELISA provides increased assay specificity and sensitivity with different sets of anti-HCP antibodies, organized into four kit types (Type A, Type C, Type D, Type E), for optimized HCP detection.
- Genetic background of the E.coli antigens: BL21 (DE3) & W3110
- Fermentation media: LB Medium & Mineral Medium
Due to the difficulties of predicting the HCP spectrum in E.coli, we recommend testing all four kit types first and then selecting the best performing kit according to ELISA key parameters, such as dilution linearity, accuracy and total coverage.
|Type||Cell line||Growth Medium|
|Type A 2G||BL21 (DE3)||Mineral Medium|
|Type E||W3110||Mineral Medium|
|Type D 2G||BL21 (DE3)||LB Medium|
|Type C||W3110||LB Medium|
According to the Annex XIV of the Regulation (EC) No 1907/2006 of REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), Triton™ X-100 (contains 4-(1,1,3,3-Tetra-methylbutyl)phenol, ethoxylated) will be replaced by TWEEN®20 (Polysorbate 20) in all our ELISA kit components by January 1st, 2021.
To read the statement of BioGenes' Management, please download this document.