Defined protein and animal component-free NS0 fed-batch culture

Abstract

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.

Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

“…Among 20 candidates, ethanolamine, putrescine, lipid mixture, sodium pyruvate, choline chloride and D-calcium pantothenate were selected as active factors for further optimization. Ethanolamine, a structural component of phospholipids in mammalian cell membrane and a precursor for numerous enzymes and cofactors (Spens and Häggström 2007), has been shown to play a positive role in promoting cell growth (Table 3), which is consistent with the previous report that ethanolamine is an essential component for hybridoma cell culture in serum-free medium (Murakami et al 1982). It was demonstrated that Fig.…”

“…In cell culture, high ammonium levels may deplete cell energy, induce apoptosis and alter glycosylation patterns of the recombinant proteins (Spens and Häggström, 2007). GS-engineered cell lines can synthesize glutamine from glutamate and ammonia, resulting in a reduction of ammonia concentration.…”

“…The feed rates of amino acids and phosphorus were determined by calculating the uptake rates of previous batch processes. For immeasurable substances including lipids, sodium selenite, ethanolamine, putrescine, vitamins and some trace elements existing in CHO-SFM, they were supposed to be depleted at the time when an increase in the death rate was observed (Spens and Häggström 2007). Then an approximately supplementing rate of each compound can be determined in the subsequent fed-batch.…”

“…In order to avoid limited nutrient concentration or excessive metabolic by-product accumulation (such as ammonia and lactate), most attempts to enhance culture productivity have focused on development of feed formulations and feed strategies (Hu et al 2011;Meghrous et al 2009;Spens and Häggström, 2007;Zhang et al 2004). Fed-batch process has become the dominant production technology for therapeutic and recombinant proteins due to its ease of operation, flexibility to be implemented and compatibility with large scale manufacturing (Huang et al 2010).…”

Metabolomics and Mammalian Cell Culture

“…There could be changes in the expression level of some proteins of the pathway due to gene regulation or other control mechanisms. In addition to the specific gene expression alterations along the cholesterol and lipid metabolism pathways, cholesterol dependence could also be the result of insufficient precursor supply (Spens and Haggstrom, 2005).…”