Monitoring cell productivity for the production of recombinant proteins by flow cytometry: An effective application using the cold capture assay

Abstract

Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically time-consuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively time-effective, and potentially cheaper alternative for monitoring the productivity of a cell culture.

Optimization of a mAb production process with regard to robustness and product quality using quality by design principles

“…The total protein concentration of the supernatant was analyzed by high-performance liquid chromatography (HPLC) as described by Meyer et al and used for calculation of the mAb peak proportions. [22] Antibody bioactivity was determined by an adherent mouse fibroblast (L929) cell based assay using the tumor necrosis factor alpha (TNF-α) under the presence of actinomycin D. [23] Therefore, L929 cell viability was analyzed using the cell titer-blue assay (Promega, USA) after 24 h of antigen/antibody treatment. The produced antibody was diluted and used in low and high concentrations of 2.5 and 50 ng/mL respectively.…”

Invitro biocompatibility evaluation of a heat‐resistant 3D printing material for use in customized cell culture devices

“…A variety of in vitro methods are available for testing cytotoxic effects, ranging from counting viable/dead cells under the microscope to biochemical assays, flow cytometric analysis, and real-time live-cell imaging technology [33,[35][36][37]. While microscopic observations -including counting of viable/dead cells using vital dyes such as trypan blue -and observations of changes in cell morphology provide an initial assessment of cytotoxic effects, biochemical assays provide more reliable and specific information [33,35,36].…”

Stress‐induced increase of monoclonal antibody production in CHO cells

“…As a result, a standardized VCC would yield a higher product titer. Previous studies showed an increase in productivity during the stationary phase and the transition phases into and out of it [19]. Importantly, the methods applied in this second approach should not impair cell growth in its exponential phase to ensure high cell concentrations during the time of highest productivity.…”