4-(2,5-Dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide improves monoclonal antibody production in a Chinese hamster ovary cell culture

Abstract

There is a continuous demand to improve monoclonal antibody production for medication supply and medical cost reduction. For over 20 years, recombinant Chinese hamster ovary cells have been used as a host in monoclonal antibody production due to robustness, high productivity and ability to produce proteins with ideal glycans. Chemical compounds, such as dimethyl sulfoxide, lithium chloride, and butyric acid, have been shown to improve monoclonal antibody production in mammalian cell cultures. In this study, we aimed to discover new chemical compounds that can improve cell-specific antibody production in recombinant Chinese hamster ovary cells. Out of the 23,227 chemicals screened in this study, 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide was found to increase monoclonal antibody production. The compound suppressed cell growth and increased both cell-specific glucose uptake rate and the amount of intracellular adenosine triphosphate during monoclonal antibody production. In addition, the compound also suppressed the galactosylation on a monoclonal antibody, which is a critical quality attribute of therapeutic monoclonal antibodies. Therefore, the compound might also be used to control the level of the galactosylation for the N-linked glycans. Further, the structure-activity relationship study revealed that 2,5-dimethylpyrrole was the most effective partial structure of 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide on monoclonal antibody production. Further structural optimization of 2,5-dimethylpyrrole derivatives could lead to improved production and quality control of monoclonal antibodies.

Fig 1. Results of the first screening to evaluate the effect of the chemical compounds on mAb production.

Higher mAb production is shown as a positive arbitrary unit.

Fig 2. Relative mAb concentration and cell-specific productivity increased with the addition of the chemical compounds.

The numbers in and above the circles indicate viability and the sample’s ID number, respectively.

Fig 3. The effect of the three candidate compounds on relative mAb concentration and relative cell-specific productivity.

All cell cultures were executed three times and evaluated on day 3. Each value of relative mAb production (A) and relative cell-specific productivity (B) were compared statistically.

Fig 4. The structure of 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide (MPPB).

Fig 5. MPPB suppresses cell growth and increases cell-specific productivity.

The tested MPPB concentrations are indicated in the figure. In these tests, viable cell density (A), viability (B), cell-specific productivity on day 4 (C), mAb concentration on day 4 (D) were evaluated.

Fig 6. MPPB increased both intracellular ATP amounts and cell-specific glucose uptake rates and suppressed cell growth.

Given previous results, the tested MPPB concentrations ranged from 0.32 to 0.64 mM. Intracellular ATP amounts (A), cell-specific glucose uptake rates (B), relative cell-specific productivity (C), and viable cell density (D) were evaluated on day 3.

Fig 7. Cell culture profile and mAb production in rCHO cells treated with MPPB.

MPPB at 0.64 mM was added in the batch cultures on day 0. Viable cell density (A) and viability (B) were measured every 2 days. Cell culture was continued until viability below 70% was reached. The slope of mAb concentration (C) to integral viable cell concentration was used to indicate cell-specific productivity (D) [28].

Fig 8. The effect of MPPB on the metabolite profile in the batch cultures.

The spent media analysis of glucose concentration (A) and lactate concentration (B) was conducted every 2 days starting on day 2. Cell-specific glucose uptake rates (C) and cell-specific lactate production rates (D) were indicated as the slopes of consumed glucose concentration and lactate concentration, respectively to integral viable cell concentration [28, 29].

Fig 9. Cell culture profile and mAb production in the fed-batch cultures under the MPPB-added condition.

MPPB at 0.64 mM was added to a culture on day 0, and 2% (v/v) feed medium was added on days 2, 4, and 6. Viable cell density (A) and viability (B) were measured every 2 days. The cell culture was continued until viability below 70% was reached. The slope of the mAb concentration (C) and integral viable cell concentration were used as cell-specific productivity (D) [28].

Fig 10. Metabolite analysis of the fed-batch cultures under the MPPB-added condition.

Glucose concentration (A) and lactate concentration (B) were measured before adding the feed medium. Glucose concentration after adding the feed medium was calculated using the added feed medium amount. Cell-specific glucose uptake rates (C) and cell-specific lactate production rates (D) were indicated as the slopes of consumed glucose concentration and lactate concentration, respectively, against integral viable cell concentration [28, 29].

Fig 11. MPPB suppresses galactosylation of mAb.

Each value was calculated as the percentage of total N-linked glycans. The ratio was statically analyzed.

Fig 12. Five chemical components derived from MPPB.

Fig 13. Evaluation of the effect of the five chemical components derived from MPPB on cell-specific productivity.

Each chemical compound was added on day 0 and evaluated on day 3. The tested concentration of each chemical compound was 0.32 mM. Lane 1: dimethyl sulfoxide (control); lane 2: MPPB; lane 3: N-(2,5-dioxopyrrolidin-1-yl) benzamide; lane 4: 4-(2,5-dimethyl-1H-pyrrol-1-yl) benzamide; lane 5: succinimide; lane 6: 4-aminobenzamide; lane 7: 2,5-dimethylpyrrole.

Fig 14. Additional pyrrole derivatives tested to identify the structure-activity relationship of MPPB with viability and relative cell-specific productivity in rCHO cell cultures.