Differential response in downstream processing of CHO cells grown under mild hypothermic conditions

Abstract

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L−1. However, host cell proteins, measured by ELISA, increased by ∼50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI-TOF MS and 2D-PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:688–696, 2013

Model‐based investigation of intracellular processes determining antibody Fc‐glycosylation under mild hypothermia

“…This is multiplied by a term that accounts for NSD utilization for both host cell protein and antibody product glycosylation, which are functions of cell growth and specific mAb productivity, respectively. The total number of glycans per cell (mmol cell −1 ) at 36.5°C was 1.38 × 10 −12 mmol cell −1 , based on the calculations from Harrison et al () and Selvarasu et al () for the average host cell weight and biomass composition (Supplementary Table SIV), but re‐estimated by the model as 2.07 × 10 −12 mmol cell −1 at 32°C which is of the same magnitude as the increase in the amount of host cell protein suggested in Tait et al (); while the number of NSD molecules consumed per host cell N‐linked glycan (mmol NSD mmol glycan −1 ) was taken from Raman et al (). The amount of NSD consumed per mAb Fc‐glycan (mmol NSD mmol glycan −1 ) was calculated from the experimentally determined Fc‐glycan compositions (Sou et al, ), while , the number of glycan chains per molecule of product (mol gly mol mAbFc −1 ), is 2 assuming that all IgG molecules produced are glycosylated (Jedrzejewski et al, ).…”

Cascading effect in bioprocessing—The impact of mild hypothermia on CHO cell behavior and host cell protein composition

“…At the same time, nutrients and energy are redistributed from biomass synthesis to production of recombinant proteins (Gammell, Barron, Kumar, & Clynes, ). The impact of cell culture temperature on HCP profile has been previously studied by Jin, Szapiel, Zhang, Hickey, and Ghose () and Tait et al (). However, these two studies produced different conclusions regarding the HCP profile at harvest.…”

“…Recent research has shown that cell culture conditions, such as cell age, harvest time, and cell culture temperature, affect HCP composition in harvested cell culture fluid (HCCF) and in purified samples (Hogwood, Bracewell, & Smales, ; Tait, Tarrant, Velez‐Suberbie, Spencer, & Bracewell, ; Valente, Lenhoff, & Lee, ). For example, Valente et al () found that when CHO‐K1 cells of different ages were cultured under the same conditions, they differentially expressed 92 HCPs, 34 of which have been reported as difficult to remove by purification, and 17 were known to strongly interact with mAbs.…”

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells

“…Indeed, achieving high yields of recombinant protein of a clinically acceptable quality is dependent on a multiplicity of parameters, including the choice of the host expression system, the achieved viable cell mass [3,4], gene copy number [5], site-specific integration [6,7], the cellular processes responsible from gene to protein in the synthesis of the desired product [1,8], the bioreactor environment (e.g. nutrients and oxygen levels) [2,9], the authenticity and homogeneity of the product [10,11] and the yield and success of downstream processing [12,13]. …”