The Chinese hamster ovary (CHO) cell line is the most prevalent biopharmaceutical expression system, and has been proven safe for the commercial production of protein therapeutics. To ensure the safety of recombinant-expressed biopharmaceuticals, health authorities are demanding the close monitoring of HCP impurities by applying HCP detection methods with high sensitivity and specificity. The CHO|360-HCP ELISA is an enhanced generic HCP assay composed of four different generic kits, which was designed to cover a broader spectrum of CHO-HCPs compared to single generic kits.
The CHO|360-HCP ELISA kits (Type A to D) are based on a unique assay design, employing differently-prepared HCP antigens and antibodies from two species:
- The antigen is prepared from total and fractionated HCP obtained from mock fermentation of CHO-K1 and CHO-S
- The specific antibodies are generated by parallel immunization of goats and rabbits
|Type A||Goat||Total HCP|
|Type B||Goat||Fractionated HCP|
|Type C||Rabbit||Total HCP|
|Type D||Rabbit||Fractionated HCP|
To show this, we tested CHO|360-HCP ELISA kits (A to D) and a commercially available generic CHO HCP assay, on the basis of a great number of mock CHO HCP samples. In most cases, the best recovery was achieved with one of the four CHO|360-HCP ELISAs kits, as compared to other commercial kits (see right-hand image).
- Sample 4: a recovery value of > 90% was estimated with ELISA kit type D while ELISA kits A, B and C and the commercial kit were less sensitive, with the latter detecting only 20% of the host cell proteins.
- Recovery values > 100% may occur due to overestimation.
Naturally, low molecular weight (LMW) proteins are less immunogenic than larger proteins, leading to a reduced production of anti-LMW HCP antibodies after the immunization of animals. Consequently, very weakly immunogenic or non-immunogenic HCPs may not be detected by ELISA or Western Blot. The BioGenes'
CHO|360-HCP ELISA solved this by using the total and fractionated HCP for the immunization of animals, resulting in four different HCP ELISA kits (A to D).
Antibodies obtained from immunization with each of three different molecular-weight fractions were then pooled again to combine antibody reactivity towards to the complete HCP spectrum. The immunization of rabbits and goats enables the generation of distinct antibody panels, which allows for a broader coverage of HCPs in customized samples.
The BioGenes recommendation:
We recommend testing all four kit types (Starter Set) first, and then selecting the best performing kit according to ELISA key parameters (such as dilution linearity, accuracy and total coverage) for use in all subsequent tests.
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.