ClonePix FL Research Articles

Optimisation of culture conditions for a producer clone coexpressing arylsulfatase B and a formylglycine-generating enzyme in order to increase the yield of arylsulfatase B

Maroteaux—Lamy syndrome (mucopolysaccharidosis type VI) is an orphan genetic disease caused by mutations in the arylsulfatase B gene (ARSB), which encodes the lysosomal enzyme arylsulfatase B (ASB). The relevance of the study lies in the need of a Russian recombinant ASB product for patients with the disease in the Russian Federation. Previously, the authors have developed producer lines coexpressing the target ASB enzyme with an auxiliary formylglycine-generating enzyme (FGE), based on Chinese hamster ovary (CHO) cells. Further development of the recombinant ASB preparation places priority on increasing the enzyme yield. The aim of this study was to increase the productivity of producer clones by optimising the culture process and adding calcium chloride and copper sulfate to the culture medium. Materials and methods: a suspension-adapted CHO cell line was used. Monoclonal cell lines were developed using Cell Metric and ClonePix FL systems. The concentration of ASB in the culture liquid was determined using the enzyme-linked immunosorbent assay (ELISA). The authors analysed batch culture and/or fed-batch culture in media supplemented with various concentrations of copper sulfate and calcium chloride. Results: the combined addition of copper sulfate and calcium chloride at concentrations of 300 μM during batch culture of producer clones coexpressing ASB and FGE increases viability and specific productivity of the cells up to 4.58±1.62 pg/ (cell×day). The cultivation of the lead producer clone coexpressing ASB and FGE under fed-batch conditions for 12 days and the addition of copper sulfate to the growth medium at the concentration of 300 μM allow for increasing the yield of the active lysosomal enzyme, arylsulfatase B, to 420 mg/L. Conclusions: the cultivation of producer clones coexpressing ASB and FGE under fed-batch conditions with copper sulfate added to the medium significantly improves cell line growth properties and the ASB yield. This approach to the selection of culture conditions for producer cell lines can be applied to other enzymes of the sulfatase family.

Sequential screening by ClonePix FL and intracellular staining facilitate isolation of high producer cell lines for monoclonal antibody manufacturing

Screening and characterization of cell lines for stable production are critical tasks in identifying suitable recombinant cell lines for the manufacture of protein therapeutics. To aid this essential function we have developed a methodology for the selection of antibody expressing cells using fluorescence based ClonePix FL colony isolation and flow cytometry analysis following intracellular staining for immunoglobulin G (IgG). Our data show that characterization of cells by flow cytometry early in the clone selection process enables the identification of cell lines with the potential for high productivity and helps to eliminate unstable cell lines. We further demonstrate a correlation between specific productivity (qP) and intracellular heavy chain (HC) content with final productivity. The unique combination of screening using ClonePix FL and the flow cytometry approaches facilitated more efficient isolation of clonal cell lines with high productivity within a 15week timeline and which can be applied across NS0 and CHO host platforms. Furthermore, in this study we describe the critical parameters for the ClonePix FL colony based selection and the associated calculations to provide an assessment of the probability of monoclonality of the resulting cell lines.

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system

Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePixFL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line.

High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system

Therapeutic recombinant monoclonal antibodies (mAbs) are commonly produced by high-expressing, clonal, mammalian cells. Creation of these clones for manufacturing remains heavily reliant on stringent selection and gene amplification, which in turn can lead to genetic instability, variable expression, product heterogeneity and prolonged development timelines. Inclusion of cis-acting ubiquitous chromatin opening elements (UCOE™) in mammalian expression vectors has been shown to improve productivity and facilitate high-level gene expression irrespective of the chromosomal integration site without lengthy gene amplification protocols. In this study we have used high-throughput robotic clone selection in combination with UCOE™ containing expression vectors to develop a rapid, streamlined approach for early-stage cell line development and isolation of high-expressing clones for mAb production using Chinese hamster ovary (CHO) cells. Our results demonstrate that it is possible to go from transfection to stable clones in only 4 weeks, while achieving specific productivities exceeding 20 pg/cell/day. Furthermore, we have used this approach to quickly screen several process-crucial parameters including IgG subtype, enhancer-promoter combination and UCOE™ length. The use of UCOE™-containing vectors in combination with automated robotic selection provides a rapid method for the selection of stable, high-expressing clones.

Strategies for clone detection, selection and isolation in Per.C6 cells - case for Rebmab100

Background A successful monoclonal antibody (mAb) cell line development requires efficient clone detection and screening. Cloning by limiting dilution (LDC) is the traditional method to isolate mAbs expressing clones [1]. Although effective, LDC is time-consuming, with limited workflow and therefore a critical step of cell line development. To compare to LDC in terms of timelines and productivities for Rebmab100 mAb cell line development we have implemented ClonePix FL (CP-FL), an automated system for high throughput clone detection. The robotic colony picker has the advantages of reducing the process time and increasing the probability to isolate highproducing clones. Moreover, we have combined these two approaches with high throughput screening assays for early detection of high productive clones. Rebmab100 mAb targets Lewis-Y, a blood group-related antigen expressed in over 70% of epithelial cancers, including breast, colon, ovary and lung carcinomas. The murine monoclonal 3S193 was generated in BALB/c mice by immunization with Le-expressing cells from the MCF-7 breast carcinoma cell line [2]. The humanized version of antiLe 3S193 mAb was obtained by CDR-grafting method [3]. The hu3S193 (Rebmab 100) mAb has potent immune effector function (ADCC and CDC), is rapidly internalized into Le expressing cancer cells, and has been shown to cause significant regressions in xenograft models in preclinical studies, alone or in conjunction with isotope and toxins [3,4]. Safety and desirable pharmacokinetic profiles of Rebmab100 were demonstrated in a Phase I clinical trial in patients with epithelial carcinomas [5] and promising results have been obtained in a Phase II clinical trial conducted in Brazil [6]. Very importantly, Rebmab100 was granted orphan-drug status by the FDA for ovary cancer. Aiming the next step of Rebmab100 mAb development we generated a new Rebmab100 cell line that shows stability and high productivity allowing its scale-up to later clinical trials.

Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity

The selection of high-producing mammalian cell lines is a crucial step in process development for the production of biopharmaceuticals. Previously, cloning by limiting dilution method was used to isolate monoclonal NS0 cells secreting high levels of humanized-C2 monoclonal antibodies. However limiting dilution method is time consuming, has low probability of monoclonality and is significantly limited by the number of clones that can be feasibly screened. In order to minimize the duration and to increase the probability of obtaining high-producing clones with high monoclonality, an automated colony picker, Clone Pix FL system was used to replace limiting dilution method. We were able to screen 1 x 10 5 clones secreting humanized monoclonal antibodies and high producer clones were selected in just 7 days. Briefly, semi-solid media was used to immobilize single cells separately and allow them to proliferate into discrete clones. The high viscosity nature of the semi-solid media retains the secreted products in the vicinity of the associated clones. Using Clone Pix FL system, all clones were screened and the producer clones with different exterior fluorescent intensities were automatically isolated. We were able to isolate rare high-producers (> 3000 FU) with frequency of as low as 0.003% of the population. A quantitative ELISA was also performed to evaluate the correlation between the fluorescence intensity of clones with its corresponding antibody productivity. Clones with fluorescence intensity of

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Stem and iPS cell selection: quantitation of surface marker (SSEA1) and intracellular GFP

Consistent and reliable propagation of undifferentiated stem cells is of particular interest in cell line development and drug screening. The Genetix ClonePix FL technology, already established for the rapid one-step identification and isolation of hybridomas and cell lines for biopharmaceutical production, can be used to isolate and collect stem cells expressing specific cell surface and intracellular proteins.

Rapid automated selection of mammalian cell colonies by cell surface protein expression

Genetix's unique ClonePix FL technology uses our ability to grow mammalian cells into clonal colonies suspended in semisolid media, and image these colonies using fluorescence assays and markers. Here we describe a new application for this technology in the isolation of clonal cell populations based on their expression of cell surface proteins.

Rapid isolation of antigen-specific clones from hybridoma fusions

The Genetix ClonePix FL is a powerful way to find and collect antigen-specific clones from hybridoma fusions in a rapid one-step process—shortening the process timeline and allowing parallel interrogation of multiple antigens in the monoclonal antibody discovery process. ClonePix FL automatically screens the entire hybridoma fusion in situ and isolates only antigen-specific clones with the desired immunoglobulin isotype. If the fusion has many antigen-positive clones, then ClonePix FL can selectively collect only the best secretors.

Rapid selection of mammalian cells secreting large amounts of therapeutic proteins or peptides

Screening cell populations for high producing clones is a key rate-limiting step in the production of protein therapeutics. The Genetix ClonePix FL provides a powerful way to find and collect the highest producing clones in a rapid one-step process. Clonal colonies of transfected CHO, HEK293 or myeloma cells secreting a recombinant protein of interest are probed in situ with target protein–specific, fluorophore-conjugated antibody, and then selected based on their protein secretion rate using robotic processing and picking techniques. This process considerably improves efficiency and streamlines traditional workflows, generating optimal cell lines that secrete the highest levels of the desired protein or peptide.