Fermentanomics informed amino acid supplementation of an antibody producing mammalian cell culture

Abstract

Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools.

Selection of chemically defined media for CHO cell fed-batch culture processes

“…In the combination of OptiCHO + EFA/B, apart from asparagine and glutamine, also tyrosine and cystine (Appendix 4 of ESM) were depleted around the culture transition point for both clones, which might be related to the lower mAb productivities (Appendix 3 of ESM). Similar observations of tyrosine and cystine depletion affecting protein production were shown for CHO cells (Yu et al 2011) and hybridoma cells (Read et al 2013). The non-essential amino acids can be produced by cells, therefore the effects of their depletion at extracellular level to the culture performance could not be confirmed in this study.…”

Bioreactor Process Parameter Screening Utilizing a Plackett-Burman Design for a Model Monoclonal Antibody

“…Unless noted otherwise in the text, reagents were purchased from the same source and used at similar concentrations as described in Read et al Chemically defined (CD) hybridoma AGT culture media (Gibco, Grand Island, NY) was prepared with supplemented amino acids (aspartate, cysteine, methionine, threonine, tryptophan, and tyrosine). Supplements used in the experimental design included: hydrocortisone (Sigma‐Aldrich, St. Louis, MO), 1× MEM non‐essential amino acids (NE‐AA) (Corning Cell Gro, Manassas, VA), and CD lipid concentrate (Gibco).…”

“…A saturated 12 experiment Plackett–Burman design was chosen to screen 11 variables at two levels (Table ). Meaningful operating ranges for all parameters were chosen based on biologically relevant ranges, commercial practices, and previous empirical run data from the same cell line and bioreactor platform. Independent variable levels were set at either a low or high value for the five continuous variables and as action or no action for six categorical variables.…”

“…Biosensors were regenerated before each column of samples was measured using 1% (v/v) acetic acid as a regeneration buffer and 50 mmol/L Tris, pH 8.7 as a neutralization buffer. Concentrations were calculated using Octet ® Analysis Software (V. 8.0, ForteBio) and a seven point standard curve (determined orthogonally by our standard UV spectrophotometry protocol) of the model IgG3 using an initial slope equation. Specific productivity (Qp) was calculated as the volume adjusted antibody yield over the IVCD.…”

Scale-down of CHO cell fed-batch cultures

“…In the combination of OptiCHO + EFA/B, apart from asparagine and glutamine, also tyrosine and cystine were depleted around the culture transition point for both clones, which might be related to the lower mAb productivities ( Table II). Similar observations of tyrosine and cystine depletion affecting protein production were shown for CHO cells 67 and hybridoma cells 68 . The non-essential amino acids can be produced by cells, therefore the effects of their depletion at extracellular level to the culture performance could not be confirmed in this study.…”