Representative mammalian cell culture test materials for assessment of primary recovery technologies: A rapid method with industrial applicability

Abstract

Mammalian cell culture material is often difficult to produce accurately and reproducibly for downstream studies. This article presents a methodology for the creation of a set of cell culture test materials where key variables including cell density, cell viability, product, and the host cell protein (HCP) load can be manipulated individually. The methodology was developed using a glutamine synthetase Chinese hamster ovary cell line cultured at 5-L and 70-L scales. Cell concentration post-cell growth was manipulated using tangential flow filtration to generate a range of target cell densities of up to 100 × 106 cells/mL. A method to prepare an apoptotic cell stock to achieve target viabilities of 40–90% is also described. In addition, a range of IgG1 and HCP concentrations was achieved. The results illustrate that the proposed methodology is able to mimic different cell culture profiles by decoupling the control of the key variables. The cell culture test materials were shown to be representative of typical cell culture feed material in terms of particle size distribution and HCP population. This provides a rapid method to create the required feeds for assessing the feasibility of primary recovery technologies designed to cope with higher cell density cultures.

Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures

“…A typical particle size distribution profile of the CY01 cell line has been previously shown, where cell debris were found in the range of 2-5 μm, cells (including apoptotic and live) >5 μm [27,30]. HCP release into the supernatant stream was also observed, indicating high levels of cell damage due to shear.…”

“…where TP is the total protein concentration and TY s is the total product concentration in the sample. IgG 1 concentration was determined using a Protein G column (HiTrap™, GE Healthcare, UK) on an HPLC system (Agilent Technologies, UK), as previously described in Popova et al . Concentration yield ( Y CP ) was calculated for the assessment of membrane performance at small scale using the following equation: …”

“…Cell culture was carried out using the CY01 cell line, kindly donated by Lonza Biologics, in 5 L STRs (B. Braun BIOSTAT B‐DCU control unit, Sartorius, Epsom, UK) and harvested on day 13. Cell culture set points have been carried out as previously described in Popova et al . Total cell concentrations at harvest were ∽9 × 10 6 cells/mL with an average viability of 77%.…”

“…Cell culture test materials (CCTM) were used for this purpose. The method for CCTM generation allowed independent control of impurity, product, cell density and viability variables to create the perceived most demanding feed material conditions . The technologies were evaluated based on the following performance criteria: solids removal, yield and impurity removal.…”

A protocol to transfer a fed‐batch platform process into semi‐perfusion mode: The benefit of automated small‐scale bioreactors compared to shake flasks as scale‐down model

“…However, the viability in both experimental setups decreased to 60% on the last two cultivation days (Supporting Information Figure S2 and Figure 4). As viabilities below 70% are common harvest criteria, 10,37,38 further improvement of the process stability and higher CSPR values are desirable.…”