Burkhard Wilms

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.

Deletion of a telomeric region on chromosome 8 correlates with higher productivity and stability of CHO cell lines

Chinese Hamster Ovary (CHO) cells are widely used for large scale production of recombinant biopharmaceuticals. Although these cells have been extensively used, a demand to further increase the performance, for example, to facilitate the process of clone selection to isolate the highest producing cell lines that maintain stability of production over time is still existing. We compared gene expression profiles of high versus low producing CHO clones to identify regulated genes which can be used as biomarkers during clone selection or for cell line engineering. We present evidence that increased production rates and cell line stability are correlated with the loss of the telomeric region of the chromosome 8. A new parental CHO cell line lacking this region was generated and its capability for protein production was assessed. The average volumetric productivity of cells after gene transfer and selection was found to be several fold improved, facilitating the supply of early drug substance material to determine for example, quality. In addition, significantly more cell clones with a higher average productivity and higher protein production stability were obtained with the new host cell line after single cell cloning. This allows reduced efforts in single cell sorting, screening of fewer clones and raises the opportunity to circumvent time and labor‐intensive stability studies. Biotechnol. Bioeng. 2016;113: 1084–1093.

Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

Chinese Hamster Ovary (CHO) cells are widely used for the large‐scale production of recombinant biopharmaceuticals. However, attempts to express IGF‐1 (a mutated human Insulin‐like growth factor 1 Ea peptide (hIGF‐1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1–0.2 g/L). Human IGF‐1 variants negatively impacted CHO cell growth via the IGF‐1 receptor (IGF‐1R). Therefore knockout (KO) of the IGF‐1R gene in two different CHO cell lines as well as knockdown (KD) of IGF‐1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF‐1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10‐fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells. Biotechnol. Bioeng. 2016;113: 1094–1101.

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.

Technology toolbox for cell line development - next generation cell line development technologies

Background Chinese hamster ovary (CHO) cells are the most widely used host for large scale production of recombinant therapeutic proteins. A combination of several gene editing approaches applied to Novartis proprietary CHO cell line resulted in a superior cell line with a significant increase of titer and improved product quality. Inter alia we have surprisingly identified a key protease responsible for proteolytic degradation of mainly non-antibody format therapeutic proteins. The recently published CHO genome in combination with screening methods and cell line engineering tools has enabled the development of this novel CHO cell line.

Generation and characterization of a superior host cell line for biomanufacturing

Background Chinese hamster ovary (CHO) cells are the most widely used host for large scale production of recombinant therapeutic proteins exhibiting high productivities in the gram per liter range. Although these cells have been extensively characterized and optimized, a demand to further increase the performance towards higher productivity and clonal stability exists. Up to date, the clone selection process is mainly based on phenotypic screens like titer measurements and growth data instead of more reliable biomarkers. Recently, the genomes of Chinese hamster as well as of different CHO cell lines were sequenced, assembled and annotated [1-3]. This new information gives the opportunity for functional analysis of the transcriptome and allows rational designs for the identification of biomarkers for clone selection and targets for cell line engineering.