Holger Laux

Expression of the human cytomegalovirus pentamer complex for vaccine use in a CHO system

Human cytomegalovirus (HCMV) causes significant disease worldwide. Multiple HCMV vaccines have been tested in man but only partial protection has been achieved. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of neutralizing antibodies in HCMV seropositive individuals and raises high titers of neutralizing antibodies in small animals and non‐human primates (NHP). Thus, Pentamer is a promising candidate for an effective HCMV vaccine. Development of a Pentamer‐based subunit vaccine requires expression of high amounts of a functional and stable complex. We describe here the development of a mammalian expression system for large scale Pentamer production. Several approaches comprising three different CHO‐originated cell lines and multiple vector as well as selection strategies were tested. Stable cell pools expressed the HCMV Pentamer at a titer of approximately 60 mg/L at laboratory scale. A FACS‐based single cell sorting approach allowed selection of a highly expressing clone producing Pentamer at the level of approximately 400 mg/L in a laboratory scale fed‐batch culture. Expression in a 50 L bioreactor led to the production of HCMV Pentamer at comparable titers indicating the feasibility of further scale‐up for manufacturing at commercial scale. The CHO‐produced HCMV Pentamer bound to a panel of human neutralizing antibodies and raised potently neutralizing immune response in mice. Thus, we have generated an expression system for the large scale production of functional HCMV Pentamer at high titers suitable for future subunit vaccine production. Biotechnol. Bioeng. 2015;112: 2505–2515.

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.

Disruption of the gene C12orf35 leads to increased productivities in recombinant CHO cell lines.

Recently, we reported that the loss of a telomeric region of chromosome 8 in Chinese Hamster Ovary (CHO) cells correlates with higher recombinant productivities. New cell lines lacking this region, called CHO‐C8DEL, showed several advantages during cell line generation and for the production of recombinant proteins (Ritter et al., 2016, Biotechnol Bioeng). Here, we performed knock‐down and knock‐out experiments of genes located within this telomeric region of chromosome 8 to identify the genes causing the observed phenotypes of CHO‐C8DEL cell lines. We present evidence that loss or reduced expression of the gene C12orf35 is responsible for higher productivities and shorter recovery times during selection pressure. These effects are mediated by increased levels of mRNA of the exogenes heavy chain (HC) and light chain (LC) as well as dihydrofolate reductase (DHFR) and neomycin phosphotransferase (Neo) during the stable expression of antibodies. Biotechnol. Bioeng. 2016;113: 2433–2442.

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.

Fam60A plays a role for production stabilities of recombinant CHO cell lines

Recombinant CHO (Chinese hamster ovary) cell lines producing therapeutic proteins often lose their production capability during long-term cultivation. To ensure that CHO production cell lines can be up-scaled to high-volume bioreactors, labor intensive stability studies of several months have to be performed to deselect clones that are losing productivity over time. The ability to predict whether clones will produce recombinant proteins at constant high levels, for example, through determination of biomarkers such as expression of specific genes, plasmid integration sites, or epigenetic patterns, or even to improve CHO host cell lines to increase the probability of the generation of stable clones would be highly beneficial. Previously, we reported that the lack of a telomeric region of chromosome 8 correlates with increased productivities and higher production stabilities of monoclonal antibody expressing CHO cell lines (Ritter A, Voedisch B, Wienberg J, Wilms B, Geisse S, Jostock T, Laux H. 2016a. Biotechnol Bioeng 113(5):1084-1093). Herein, we describe that the knock-out of the gene Fam60A, which is one of the genes located within the telomeric region of chromosome 8, in CHO-K1a cells leads to the isolation of significantly more clones with higher protein production stabilities of monoclonal antibodies during long-term cultivation. Biotechnol. Bioeng. 2017;114: 701-704.

Degradation of recombinant proteins by Chinese hamster ovary host cell proteases is prevented by matriptase-1 knockout: LAUX et al.

An increasing number of nonantibody format proteins are entering clinical development. However, one of the major hurdles for the production of nonantibody glycoproteins is host cell–related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics, and genetic knockdowns, we have identified, out of the >700 known proteases in rodents, matriptase‐1 as the major protease involved in the degradation of recombinant proteins expressed in CHO‐K1 cells. Subsequently, matriptase‐1 was deleted in CHO‐K1 cells using “transcription activator‐like effector nucleases” (TALENs) as well as zinc‐finger nucleases (ZFNs). This resulted in a superior CHO‐K1 matriptase (KO) cell line with strongly reduced or no proteolytic degradation activity toward a panel of recombinantly expressed proteins. The matriptase KO cell line was evaluated in spike‐in experiments and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next‐generation sequencing screening methods, and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins.

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.