Hans-Peter Knopf

Combination of the 2A/furin technology with an animal component free cell line development platform process

The recently described 2A/furin technology combines both chains of the antibody in a single open reading frame. Upon translation and secretion, the peptide is processed by the cell to generate native fully functional IgG antibodies. Here, we describe the results of an evaluation study of this technology for an industrial CHO cell line development process. The 2A/furin expression cassette setup was combined with a Novartis vector system. A transfection, selection, and cloning procedure in chemically defined media was established at Novartis and applied for a monoclonal test antibody. The productivity of 2A/furin-vector-derived clones in non-optimized generic shake flask fed-batch models was in a comparable range with clones derived from the reference control vector. Higher clonal production stability was seen for the majority of clones generated with the 2A/furin technology compared to the clones generated with the reference control vector. Product quality was analyzed by SDS-PAGE and no significant difference was detected between the two systems. Thus, it was shown that the 2A/furin technology can be successfully combined with a Novartis CHO expression system and platform. Due to the single ORF setup, the 2A/furin technology may therefore offer a suitable approach to reduce vector size and complexity.

Mammalian stable expression of biotherapeutics.

Many therapeutically relevant proteins, like IgG antibodies, are highly complex, multimeric glycoproteins that are difficult to express in microbial systems and thus usually produced in mammalian host cells. During the past two decades, stable mammalian expression technologies have made huge progress resulting in highly increased speed of cell line development and yield of manufacturing processes. Here, we give an overview of technologies that are applied at different stages of state-of-the-art cell line development processes for biomanufacturing.

Surface display vectors for selective detection and isolation of high level antibody producing cells

Cell line generation for production of biopharmaceuticals in mammalian cells usually involves intensive screening of clones to identify the rare high producers. In order to facilitate efficient and selective fluorescence activated cell sorting (FACS) based enrichment and cloning of antibody producing CHO cells, we developed a special vector setup by inserting a leaky translation termination signal between the heavy chain of an IgG antibody and an IgG transmembrane domain. Partial read‐through during translation of the antibody heavy chain leads to display of a subset of the produced antibody on the surface of the expressing cell. We could show that the level of surface expression correlates well with the productivity. By applying FACS, high producing cells can be selectively enriched and cloned. Two sequential FACS enrichment cycles were performed which led to more than eightfold increased productivities of transfected and selected cell populations without cloning. The combination of selective FACS enrichment and FACS cloning with the new vector setup led to a sevenfold higher average productivity of the resulting clones as compared to a reference vector. Productivity and production stability assessment of clones generated with the new vector showed no negative impact of the co‐expression of transmembrane antibody. Clone productivities of 4 g/L in a generic shake flask fed‐batch model were achieved. Thus, this new vector setup facilitates fast and selective isolation of high producing production cell lines and allows significant reduction of clone screening efforts during cell line development for production cell lines. Additionally, the high productivity of FACS‐enriched but non‐clonal cell populations supports rapid, high yield, and cost efficient material production in early project phases. Biotechnol. Bioeng. 2016;113: 2386–2393.