Differential gene expression of a feed-spiked super-producing CHO cell line

Abstract

Feed supplements are concentrated cell culture media that contain a variety of nutrients, which can be added during a bioprocess. During fed-batch cultivation, feed media are typically added to a growing cell culture to maximize cell and product concentrations. In this study, only a single shot of feed medium was added on day 0 to a basal cell culture medium and compared to non-supplemented basal medium (feed-spiked at day 0 versus control experiments) by cultivation of a recombinant mAb expressing CHO cell line in batch mode under controlled conditions in a bioreactor. Since the feed-spike at day 0 was based on existing medium components without introducing additional supplements, a desirable process with decreased complexity was generated. Unlike cells in basal medium, feed-spiked cultures reached almost 2× higher peak cell concentrations (10 × 10 c/mL vs. 18 × 10 c/mL) and 3× higher antibody concentrations (0.8 g/L vs. 2.4 g/L). Batch process time and the integral over the viable cell count were similar for both process types. Constantly high cell-specific production rates in feed-spiked cultures (70 pg/cell/day) compared to continuously declining rates in basal medium (from 70 to 10 pg/cell/day) were responsible for an overall 70% higher cell-specific production rate and the higher product concentrations. To associate gene expression patterns to different process proceedings, transcriptome analysis was performed using microarrays. Several transcripts that are involved with glutamine de novo synthesis and citric acid cycle were significantly upregulated on several days in feed-spiked cultures. The top identified gene ontology (GO) terms related well to cell cycle and primary metabolism, cellular division as well as nucleobase formation or regulation, which indicated a more active proliferative state for feed-spiked cultures. KEGG biochemical pathway analysis and Gene set enrichment analysis (GSEA) further confirmed these findings from a complementary perspective. Moreover, several interesting gene targets, which have not yet been associated with recombinant protein expression, were identified that related to a higher proliferative state, growth, protein synthesis, cell-size control, metabolism, cell survival as well as genes that are associated with the control of the mammalian target of rapamycin (mTOR) in feed-spiked cultures. Analysis of critical product quality attributes (i.e. glycosylation, charge variants and size distribution) showed that feed-spiking did not change antibody quality.

Hydrocyclones as cell retention device for CHO perfusion processes in single‐use bioreactors

“…Antibody titers determined in runs #2 and #3, which were carried out with the same CHO cell line, presented a similar profile to the respective viable cell density profiles, suggesting growth‐associated mAb production. In run #2, the cell‐specific productivity (q P ) increased after Day 18, that is, after the basal medium was replaced by a fortified formulation that boosted production rates by 70% (Figure 5f), which is in agreement with Reinhart et al (2018) for the same medium composition. IgG titer was monitored both in the bioreactor and in the harvest, and no product retention was observed, as expected for a HC (Figure 5e,f).…”

“…Higher cell concentrations were achieved in runs #2 and #3 compared with run#1, as illustrated in Figures 5a and 6a. In run #2, basal medium ActiPro was used as perfusion medium between Day 3 and 17, and then replaced with a fortified medium (Reinhart, Damjanovic, Castan, Ernst, & Kunert, 2018) to allow an increase of cell density in the bioreactor. However, differently from all previous experiments that used a 3/8” (9.6 mm) tubing to feed the HC, a double‐Y tubing with 3.2 mm ID (1/8”) was the only available option in the PRO bag model to connect the HC feed line.…”

Rapid development of clone‐specific, high‐performing perfusion media from established feed supplements

“…CB7a was additionally tested in CDM4NS0 as it was initially developed as a combination feed supplement with CB7b. Beneficial nutrient supplementation was demonstrated for this system in fed‐batch . With the chosen screening design at resolution III (DoE1) possible synergistic effects and two‐factor interactions cannot be resolved and were further investigated in the subsequent optimization design (DoE2).…”

“…Feed and harvest flow were controlled gravimetrically and pH was adjusted by CO 2 or 8% sodium bicarbonate. Product quality defined by size‐, charge‐, and glycan profiles was analyzed as reported in Reinhart et al…”

“…Beneficial nutrient supplementation was demonstrated for this system in fed-batch. 9 With the chosen screening design at resolution III (DoE1) possible synergistic effects and two-factor interactions cannot be resolved and were further investigated in the subsequent optimization design (DoE2). In contrast, enrichment of the basal media with all feed supplements at half-maximum and maximum levels maintained cell viabilities above 80% for 11 days (Figure 3b,d).…”