Date: 22nd April 2020
Biological therapeutics use animal cells as in vitro substrates and play a critical role in fighting many diseases such as cancers, autoimmune and genetic disorders. Host cell protein (HCP) impurities are generated by the host organism during bioproduction and can potentially reduce drug efficacy and cause adverse immunological effects. Now scientists have disrupted multiple genes to reduce HCPs resulting in higher productivity and higher purity of proteins thereby facilitating affordable high-value biopharmaceuticals.
HCPs are released or secreted from dead and viable cells and accumulate extracellularly during mammalian culture. They must be reduced to low levels (<1–100 ppm) in all cell-derived protein biotherapeutic before final product formulation. This downstream processing is costly and can represent up to 80% of the entire production costs, for example, in monoclonal antibody manufacturing.
With over 70% of protein therapeutics made in Chinese hamster ovary (CHO) cells – an epithelial cell line – scientists led by Nathan Lewis from the University of Calfornia, US, and collaborators from the Technical University of Denmark, Denmark, have created a “clean” CHO cell to eliminate unnecessary HCPs.
Bioenergetic investment on the secretome
Using systems biology computational models the team showed that, theoretically, the removal of multiple unnecessary HCPs could free up cellular resources and secretory capacity, in addition to improving product quality. Then, using proteomics, the team identified 14 target HCPs to be deleted which fulfilled at least one of three criteria: proteins that are abundant in harvested cell culture fluid (HCCF); proteins that are difficult to remove during downstream processing, or proteins having a negative impact on product quality. Using multiplex CRISPR-cas9 the 14 genes coding for the proteins were then disrupted by gene knockout (KO).
CRISPR-Cas9-mediated multiplex gene disruption
The cells were subjected in total to 4 cycles of CRISPR-Cas9-mediated gene disruption to create three cell lines containing serially stacked gene knockouts (KO) of 6, 11, and 14 genes (6xKO, 11xKO and 14xKO, respectively).
The cell density and viability of knockout cell lines were improved over wild type (WT) and showed a remarkable reduction in HCP content. Whilst the 11x and 14xKO behaved similarly, the 6xKO behaved as an intermediary in most of the subsequent tests. Total HCP was reduced in all three KOs (up to 70%) and specific HCP productivity was found to be reduced by 27%, 58% and 59% for the 6xKO, 11xKO, and 14xKO cell lines, respectively, when compared to the wild type (WT).
KOs displayed less HCP content and increased antibody production
To determine whether monoclonal antibody (mAb) production was altered in the KO cells, the team established stable pools from the KO cell lines expressing mAb Rituximab (Rit). Whilst the total protein content of the HCCF obtained from the knockout cell lines was reduced by ~50%, mAb production was actually higher in the KO cell lines. This was measured in two ways, antibody staining efficiency and Rit titer. An increase in mAb staining efficiency of 12% versus 2% for 14xKo compared with WT was seen, and a Rit titer increased from 2mg L-1 for WT to ~5 mg L-1 for 11xKO was measured.
However, further selection of high-producing clones, revealed that 14xKO cell line displayed a low single-cell survival rate and no clones could be selected that displayed sufficiently high mAb productivity, this lines therefore was excluded from further experiments.
To then determine the behaviour of clones in different media and to generate enough material for downstream processing the team used two different bioreactors. In the 6xKO and 11xKO lines, there was a substantial improvement in the product purity of Rit of 7-fold and 2-fold, respectively (calculated using the mAb/HCP ratio). Subsequent purification of the Rit mAb using various chromatography techniques then demonstrated that products from both cell lines were pure with HCPS at very low levels of 18ppm and 3ppm, respectively (compared to WT levels of 45ppm).
Furthermore, the HCP-reduced phenotype was stable over many generations and was observed in multiple clones. Finally the team showed that the resulting antibody was bioactive, suggesting the product had not been comprised by the HCP-reduction phenotype.
Conclusions and future applications:
The team here, have engineered ‘clean’ CHO cells that exhibit a substantial reduction in HPCs, and increase in protein purity. The reduced HCP content facilitated the purification of a monoclonal antibody, which will ultimately lead to decreased costs associated with downstream purification processes.
It is hoped that by further optimisation, and perhaps by combining this with other genetic modifications and bespoke engineering of the recombinant proteins that the system will improve yet further such that at least some of the downstream purification steps can be eliminated. The study highlights that large-scale genome editing in mammalian cells can continue to drive improvements throughout the bioproduction process.
Kol, S., D. Ley, T. Wulff, M. Decker, J. Arnsdorf, S. Schoffelen, A. H. Hansen, T. L. Jensen, J. M. Gutierrez, A. W. T. Chiang, H. O. Masson, B. O. Palsson, B. G. Voldborg, L. E. Pedersen, H. F. Kildegaard, G. M. Lee and N. E. Lewis (2020). “Multiplex secretome engineering enhances recombinant protein production and purity.” Nature Communications 11(1): 1908.
https://doi.org/10.1038/s41467-020-15866-w
