Controlling the time evolution of mAb N‐linked glycosylation, Part I: Microbioreactor experiments

Abstract

N-linked glycosylation is of key importance for the efficacy of many biotherapeutic proteins such as monoclonal antibodies (mAbs). Media components and cell culture conditions have been shown to significantly affect N-linked glycosylation during the production of glycoproteins using mammalian cell fed-batch cultures. These parameters inevitably change in modern industrial processes with concentrated feed additions and cell densities beyond 2 × 10 cells/mL. In order to control the time-dependent changes of protein glycosylation, an automated microbioreactor system was used to investigate the effects of culture pH, ammonia, galactose, and manganese chloride supplementation on nucleotide sugars as well as mAb N-linked glycosylation in a time-dependent way. Two different strategies comprising of a single shift of culture conditions as well as multiple media supplementations along the culture duration were applied to obtain changing and constant glycosylation profiles. The different feeding approaches enabled constant glycosylation patterns throughout the entire culture duration at different levels. By modulating the time evolution of the mAb glycan pattern, not only the endpoint but also the ratios between different glycosylation structures could be modified. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1123-1134, 2016.

Process performance and product quality in an integrated continuous antibody production process

“…The yield of antibody (Equation S1) in the harvest stream accounting for the loss of product in the bleed stream is shown in Figure C. In comparison, fed‐batch experiments using the identical media platform and cell line resulted in titers of 2–3 g/L at maximum cell densities of 20 × 10 6 cells/mL over 17 days (Villiger et al, ). The cell free harvest stream of the perfusion was sent to the intermediate surge tank with a constant flow rate of 1 RV/day.…”

Predictive glycoengineering of biosimilars using a Markov chain glycosylation model

“…Also, glycosylation reactions are not perfectly independent since they all rely upon a shared sugar nucleotide metabolic network. Future advanced models could take this into account by integrating sugar metabolism into the prediction of glycoforms, as already demonstrated for kinetic glycosylation models . Thus, efforts that employ the simplest strategy resulting in a glycoprofile that meets requirements of safety, efficacy, and similarity are preferred.…”

“…These perturbations can include various experimental techniques that can either involve cell engineering (knock‐downs or overexpression of glycosylation genes) or bioprocess control (media supplementation with nutrients or inhibitors). Although this and other glycosylation models allow one to make a priori predictions of glycoprofiles, there is still a great need for easy‐to‐use computational approaches that would directly address glycoengineering efforts and help to quantitatively predict optimal perturbation strategies to recapitulate a desired glycoprofile.…”

Sequential Multivariate Cell Culture Modeling at Multiple Scales Supports Systematic Shaping of a Monoclonal Antibody Toward a Quality Target

“…For this purpose, an extensive number of operating conditions have to be investigated. Automated high throughput experimental systems such as DWP and AMBR bioreactors have enabled the screening of tens to hundreds of process parameters simultaneously . Additionally, improvements in the process analytical technologies have been of great benefit for the efficiency and efficacy in bioprocess development.…”