Using Titer and Titer Normalized to Confluence Are Complementary Strategies for Obtaining Chinese Hamster Ovary Cell Lines with High Volumetric Productivity of Etanercept

Abstract

The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements.

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

“…3A) [41]. However, untransfected CHO-S WT cells were also able to recover up to the highest MSX-concentration of 50 μM, which is in accordance to previous work [36]. As shown in the killing curve of the untransfected 10x KO cells, the advantage of the Glul-KO system here seems to be the elimination of untransfected cells.…”

“…Plasmids for co-expression of A1AT/C1INH and ST6GAL1 were constructed with uracilspecific excision reagent cloning method as previously described [36,37] (Suppl. Fig.…”

Multiplex secretome engineering enhances recombinant protein production and purity

“…To enrich for clones with high productivity, immunostaining of Rituximab on the plasma membrane (surface staining) was performed on the Rituximab expressing cell pool by staining for 30 min at 4°C with 5 µg mL −1 anti-human IgG antibody conjugated to FITC (Invitrogen, Carlsbad, CA, USA) and subsequent single-cell sorting using fluorescence activated cell sorting (FACS) and expansion. To select clones with the highest productivity, we used the titer-toconfluency method 44 . Confluency was determined on a Celigo cytometer (Nexcelom Bioscience, Lawrence, MA, USA) and titer was determined using biolayer interferometry.…”

Multiplex secretome engineering enhances recombinant protein production and purity

“…To enrich for clones with high productivity, immunostaining of Rituximab on the plasma membrane (surface staining) was performed on the Rituximab expressing cell pool by staining with an anti-human IgG antibody conjugated to FITC (Invitrogen, Carlsbad, CA, USA) and subsequent single-cell sorting using fluorescence activated cell sorting (FACS) and expansion. To select clones with the highest productivity, we used the titer-to-confluency method 18 . Confluency was determined on a Celigo cytometer (Nexcelom Bioscience, Lawrence, MA, USA) and titer was determined using biolayer interferometry.…”