Metabolite profiling of recombinant CHO cells: Designing tailored feeding regimes that enhance recombinant antibody production

Abstract

Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the balance of intracellular and extracellular metabolites during the production process of a CHO cell line expressing a recombinant IgG4 antibody. Using this metabolite profiling approach, it was possible to identify nutrient limitations, which acted as bottlenecks for antibody production, and subsequently develop a simple feeding regime to relieve these metabolic bottlenecks. This metabolite profiling-based strategy was used to design a targeted, low cost nutrient feed that increased cell biomass by 35% and doubled the antibody titer. This approach, with the potential for utilization in non-specialized laboratories, can be applied universally to the optimization of production of commercially important biopharmaceuticals.

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

“…LC was performed by a hydrophilic interaction chromatography (HILIC) on a zwitterionic column with polymer matrix (ZIC pHILIC, 5 µm polymeric, PEEK 150 × 2.1 mm, SeQuant). Matrix effects and potential metabolite degradation were compensated by adding internal standards represented by commercially available uniformly 13 C-labeled algae extracts (Sigma Aldrich, 487945-1G) to the thawed samples before further preparation for LC-MS measurements. The internal standard contained the 13 C species of all analyzed metabolites.…”

“…Matrix effects and potential metabolite degradation were compensated by adding internal standards represented by commercially available uniformly 13 C-labeled algae extracts (Sigma Aldrich, 487945-1G) to the thawed samples before further preparation for LC-MS measurements. The internal standard contained the 13 C species of all analyzed metabolites. The quantification was done by performing an external calibration based on the 12 C/ 13 C-ratios for each metabolite.…”

“…Whole cells are quenched in ice-cold methanol [13], spun down in ice-cold phosphate buffer or saline solution [14], simultaneously filtered and quenched [15] or exposed to heat [16]. In addition to the well-known problems of leakage [17,18], temperature induced degradation of metabolites or interactions with filter materials [19], these protocols only yield averaged, cellular metabolite levels.…”

A Cyclin Distributed Cell Cycle Model in GS-NS0

“…The dead cell population (X D , Equation 12) is calculated by accounting for the specific death rate (k d ) of the viable cells and the lysis specific rate (k lys ) of the dead cells. The specific death rate (Equation 13) is linked to the extracellular substrate concentrations, particularly glutamate as this is an essential amino acid for the GS system [50]. The glutamate affinity death parameter (k dGlu ) and the exponential factor (q), define the sensitivity of the cell death to glutamate depletion.…”

“…Recently, metabolic flux analysis (MFA) has become a key tool for the study of mammalian cell cultures aiming at improving productivity and product quality. These studies [11][12][13][14] provide valuable insight on cell behaviour and assist in understanding cell metabolism. However, they neglect the intrinsic heterogeneity (e.g.…”