Establishment of a mammalian cell line suitable for industrial production of recombinant protein using mutations induced by high-energy beam radiation

Abstract

Mammalian cells are extensively used for production of biopharmaceuticals. Most cells used in industry have infinite proliferative capacity, which provides a high number of cells and corresponding productivity. However, infinite cells will continue to multiply even after cell density reaches sufficient levels. This excess proliferation aggravates the culture environment and induces low productivity. Therefore, after cell density reaches sufficient levels, downregulation of proliferation would prevent such aggravation and extend the culture period and improve productivity. To realize such suitable proliferation, we aimed to establish a novel cell line whose proliferation was spontaneously downregulated after reaching a sufficient population level. Mutagenesis using high-energy beam irradiation was used. CHO-DP12 cells were irradiated with 2.5 Gy X-rays and screened with hydroxyurea and 5-fluorouracil to eliminate any cells multiplying after confluence and to concentrate desired mutants. One clone was established and named CHO-M1. Cell cycle analysis indicated that CHO-M1 cells had a similar cell cycle profile in the exponential growth phase, but cells rapidly accumulated in G1 phase just before confluence and did not progress through the cell cycle. This suggested that until confluence, proliferation of CHO-M1 was similar to parental CHO, but after confluence, it was inhibited and under G1 arrest. The specific antibody production rate of CHO-M1 was kept high, even after confluence, while that of parental CHO was drastically decreased in stationary phase. These results suggest that the desired cell line was successfully established and that high-energy beam irradiation could be an efficient mutagenic technique for breeding industrial cells.

A Novel Method to Gently Mix and Uniformly Suspend Particulates for Automated Assays

“…4A,B and Tables 1-3). The number of CHO cells increased from 4450 seeding cells/well to 8190 cells/well after 24 h of culture, which is consistent with the 24 h doubling time reported by Chida et al, 17 indicating that the CHO cells were healthy and not damaged by PRF mixing. Supplemental Figure S17 is a photomicrograph of normalappearing CHO monolayers from microplate 1 after 24 h of incubation.…”

“…The cells had nearly doubled to 8190 cells/well from the seeding number of 4450 CHO cells. Chida et al 17 have reported that the doubling time for CHO cells is reported to be ~24 h. The standard deviation between all 384 wells was 278 cells, with a coefficient of variation of 3.9%. A photomicrograph of the well monolayers illustrated a normal morphology (Suppl.…”