Short‐ and long‐term effects on mAb‐producing CHO cell lines after cryopreservation

Abstract

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Q ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Q at increased cell age: 17 of the 24 cell lines displayed ≥20% Q decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Q decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Q decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.

Evaluation of the damage caused by in vitro culture and cryopreservation to dermal fibroblasts derived from jaguars: An approach to conservation through biobanks

“…In general, the nutrient conditions of the culture and environmental conditions promote cell establishment and cell growth. These conditions, when optimized, can allow cells to grow independent of the duration of culture, temperature reduction protocols, and the presence of cryoprotectants (Subramanian et al, 2018). Probably, the addition of serum to the culture medium has a significant impact on cell growth.…”

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

“…There are many examples where researchers have shown that increased cell age of CHO cell lines correlated with reduced expression levels of a recombinant protein 64–72 . This reduction in expression with cell age has been attributed to a number of factors including gene silencing and loss of gene copy number 73,74 but not to cryopreservation 8 . Interestingly, in these examples, many CHO cell lines were derived from random gene integration followed by single‐cell cloning, including our prior experience with cell lines generated by random integration 8 .…”

Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase‐mediated cassette exchange system

“…Percent viability and viable cell count were determined using the Vi‐Cell XR instrument (Beckman Coulter, Beverly, Massachusetts) to calculate the integrated viable cell concentration (IVCC). Cell specific productivity, Qp, was calculated in units of pg/cell/day using the slop of the linear fit obtained from plotting mAb titer versus IVCC in the production cultures based on the relationship: Titer = Qp × IVCC as described previously 33 …”

“…Cell specific productivity, Qp, was calculated in units of pg/cell/day using the slop of the linear fit obtained from plotting mAb titer versus IVCC in the production cultures based on the relationship: Titer = Qp × IVCC as described previously. 33…”