Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture

Abstract

Without a scale‐down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high‐capacity microscale system that is typically used in fed‐batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 106 mg/cell/day and 7 × 10 7 cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.

Red cell manufacturing using parallel stirred‐tank bioreactors at the final stages of differentiation enhances reticulocyte maturation

“…The rpms of 300, 400, 500, and 600 are equivalent to impeller tip speeds of 0.18, 0.24, 0.30, and 0.36 m/s, respectively, in the ambr® bioreactor. More information is available at the manufacturer's website and articles (Bresnick et al, 2018; Goldrick et al, 2017; Sewell et al, 2019). Viable cell density (VCD), viability, and cell diameter were measured using a Vi‐Cell XR (Beckman Coulter), which measures 4000–5500 cells per each counting using a trypan blue dye exclusion method.…”

Bioprocess considerations for T‐cell therapy: Investigating the impact of agitation, dissolved oxygen, and pH on T‐cell expansion and differentiation

“…Furthermore, a translation of the perfusion‐mimic procedure may not be feasible at the large scale. At scale, additional cell separation equipment based on either spinning membrane filtration, alternating tangential flow, or acoustic separation could be employed in conjunction with the bioreactors, as scalability of the perfusion‐mimic procedure has been demonstrated previously (Kelly et al, 2018; Klarer, Marsh, Ranade, & Smith, 2017; Kreye et al, 2019; Sewell et al, 2019).…”

“…The supernatant that was extracted as part of the medium exchange was analysed for nutrients and metabolites using a Bioprofile FLEX (Nova Biomedical, Waltham, MA). The viable cell number and metabolite data were then used to calculate the specific metabolite consumption rates as described by Sewell et al (2019). Equation (1) was used to calculate specific rates during the perfusion mimic cultivations: where is the concentration of the nutrient or metabolite in the cultivation broth at , is the concentration of the nutrient or metabolite in the cultivation broth at , is the concentration of the nutrient or metabolite in the medium, and is the dilution rate.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…Shake tubes (ST) were also established as an important scaledown tool for mammalian perfusion cell cultures in combination with bench-top bioreactors, giving high productivity (23 pg/cell•day) and low product loss in the bleed (around 10%) (Wolf et al, 2019b). A high-capacity microscale system for perfusion culture using in-situ gravity settling and automated sampling was studied, and the suitability of this platform for the evaluation of the performance of cell lines and the effects of process parameters for perfusion cultures were demonstrated (Sewell et al, 2019).…”