Directed evolution of mammalian anti-apoptosis proteins by somatic hypermutation.

Recently, researchers have created novel fluorescent proteins by harnessing the somatic hypermutation ability of B cells. In this study, we examined if this approach could be used to evolve a non-fluorescent protein, namely the anti-apoptosis protein Bcl-x(L), using the Ramos B-cell line. After demonstrating that Ramos cells were capable of mutating a heterologous bcl-x(L) transgene, the cells were exposed to multiple rounds of the chemical apoptosis inducer staurosporine followed by rounds of recovery in fresh medium.

Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells.

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-x(L) potently inhibit apoptosis, no studies have examined the use of the Bcl-2 family protein, Mcl-1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl-1 protein and a Mcl-1 mutant protein that is not degraded by the proteasome in a serum-free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody.

Protein and genome evolution in Mammalian cells for biotechnology applications.

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming.

E2F-1 overexpression increases viable cell density in batch cultures of Chinese hamster ovary cells.

The cell density is an inherent constraint in commercial mammalian cell cultures. Here, we describe a cell engineering strategy utilizing the overexpression of the E2F-1 cell cycle transcription factor in CHO DG44 cells that produce a monoclonal antibody in serum-free, suspension culture. Stable pools and cell lines expressing E2F-1 were isolated that attained viable cell densities 20% higher than control cell lines and continued proliferation for an additional day in batch culture.

Enhancement of transient gene expression and culture viability using Chinese hamster ovary cells overexpressing Bcl-x(L).

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells.

Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering.

Production of complex recombinant proteins requires the culture of mammalian cells in bioreactors. Inherent in these cultures is the problem of cell death, which can result from nutrient depletion, byproduct accumulation, and other bioreactor stresses which signal the cell to die through apoptosis, or programmed cell death. Apoptosis is a highly regulated pathway of both pro- and anti-apoptotic proteins that promote cell survival or death, and cell engineering efforts to inhibit the apoptosis pathway have led to increased culture viability and recombinant protein production.

Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells.

Enhanced product yields, reduction in throughput time, improved cost-effectiveness and product quality are examples of benefits gained by delaying apoptotic cell death in bioreactors. To examine the effect on recombinant protein production, bcl-x(L) was overexpressed in a CHO cell line secreting humanized monoclonal antibody directed against the alpha1beta1 integrin. When cell lines overexpressing bcl-x(L) were compared to the parent, cell viability was increased by 20% and titers by 80%.