【GS系统】GS Gene Expression System.doc

GS Gene Expression SystemPrinciples of the GS Gene Expression System Glutamine synthetase (GS) is the enzyme responsible for the biosynthesis of glutamine from glutamate and ammonium. This enzymatic reaction is the pathway for glutamine formation in a mammalian cell. In the absence of glutamine in the growth medium, the GS enzyme plays an essential role in the survival of mammalian cells in culture. Some mammalian cell lines, such as mouse myeloma lines, do not express sufficient GS to survive without added glutamine. With these cell lines, a transfected GS gene can function as a selectable marker by permitting growth in a glutamine-free medium. Other cell lines, such as Chinese hamster ovary (CHO) cell lines, express sufficient GS to survive without exogenous glutamine. In these cases, the GS inhibitor, methionine sulphoximine, can be used to inhibit endogenous GS activity such that only transfectants with additional GS activity can survive. A Complete Mammalian Expression System Lonza has developed a highly efficient mammalian expression system which makes use of selection, via glutamine metabolism, and powerful viral promoters. The GS System is well established with over 85 companies using the technology worldwide. Currently there are over 50 products in clinical trials and 5 products in-market that use the GS System. Productivity The high manufacturing cost of protein products, particularly for therapeutic proteins, means that maximising volumetric productivity is essential if economic processes are to be established. Use of the GS System reliably results in high-producing cell lines early in cell line construction programmes. Maximum expression levels attainable depend on the product but cell lines producing over 5 g/L of recombinant antibody have been created, with specific production rates in the range 15-65 pg/cell/day. Productivity in chemically defined, animal compotent-free GS-CHO bioreactor process Productivity in chemically defined, animal compotent-free GS-NS0 bioreactor process SpeedSpeed at the development stage of any biotechnology project is crucial to success. The GS System allows for rapid selection of high-producing cell lines. Product concentrations can be increased further through media and process optimization. This significantly reduces the time required to generate a cell line suitable for cGMP manufacture. Construction of full length antibodies with human constant regions is rapid and easy using Lonzas GS constant region vectors which enable the addition of any variable region in a single ligation step. Lonzas proprietary CHOK1SV host cell line is pre-adapted to growth in chemically defined animal component free (CDACF) media and suspension culture. Its pre-adaptation to suspension culture aids adaptation of GS cell lines back to growth in suspension culture after transfection. Timeline of steps performed to select high-yielding uncloned GS-CHO cell lines adapted to chemically defined, animal component-free media StabilityGS cell lines that are consistent in terms of product concentration, growth and product characteristics can be easily created. For unamplified, GS negative cell lines, such as NS0, the selective agent and inhibitor of GS activity methionine sulphoximine is not required for stability. For unamplified, GS positive cell lines, such as CHO, methionine sulphoximine is typically required to maintain volumetric productivity at a consistent level. FamiliarityThe GS System is successfully used by over 85 global pharmaceutical and biotechnology companies and by academic laboratories leading to a large body of published information that new users can draw upon. In addition, Lonza has accumulated a considerable amount of expertise, which is shared with users. Currently there are over 50 products in clinical trials and 5 products in-market that use the GS System, including Zenapax (Roche) and Synagis (Medimmune) so the system is well known to all regulatory authorities. Extensive ExperienceLonza has itself created hundreds of cell lines using the GS System. Many of these cell lines have been grown at large scale and have produced product for use in clinical trials and for in market supply. Other GS users have accumulated experience with a diverse range of products. In most cases high harvest product concentrations were achieved. Many investigators use GS, not only to develop a manufacturing process, but as a tool to create recombinant proteins for biological studies. In these cases the reliability of GS, as a consistent means of generating high-producing cell lines quickly, is invaluable.The GS System can be easily accessed by contacting the GS team at GSL and obtaining a GS Research Evaluation Agreement.GS Research Evaluation Agreement (GS REA) A GS REA allows the user to carry out a pre-clinical evaluation of the GS System with an unlimited number of products. Standard GS System PackageThe current standard package of GS System materials to which the user has access with a GS REA are: GS vectors and sequence data Vials of GMP host cell bank NS0 Vials of GMP host cell bank CHOK1SV Transfection medium system Comprehensive technical operating manuals Cell bank characterization reports to support regulatory submissions Access to technical support from Lonzas experts in the GS System via the GSL mailbox. GS newsletters (twice a year) Technology updates In house or on-site training Visits to customer sites as part of GS System tours Technical teleconference following 3 months of using the GS SystemOptional GS System PackagesGS System customers can augment their standard GS REA package by purchasing other proprietary components together with associated know-how. The current optional packages of GS System materials that users can access are: Constant region vectors pConPlus vectors Serum-free medium, supplements and feed systems (serum-free packages) Chemically defined animal component free medium, supplements and feed systems (protein-free packages) Host cell protein Western blotting assays Host cell protein ELISA assays Methionine sulphoximine assaysGS License AgreementGS licenses can also be obtained to allow clinical development and commercialization of a product or products produced using the GS System. However, users will often continue to hold a GS REA, even with a GS license, to allow for continued evaluation of new products in the laboratory. ServicesIn addition, Lonza can create and select GS cell lines as a service for customers and can manufacture cGMP product if required. The advantage of this approach is that Lonza brings years of accumulated GS experience to the project. Contact To access the GS System, please contact: GSL, Tel +44 1753 777 000High Yielding Cell Culture Media and Feed Platform As a leader in technology and innovation for mammalian cell culture, Lonza has developed a new version of their cell culture media and feed platforms for the GS Gene Expression System. The new offering includes Lonzas proprietary chemically defined animal component free (CDACF) version 8 media and feeds system which was designed in conjunction with the GS-CHO cell line. The new version 8 platform incorporates GS-CHO specific CDACF cell culture medium, feed base powders and supplements and is globally manufactured and supplied by Lonzas custom media (hyperlink to the custom media page) production facilities. The CDACF version 8 system has been shown to reach yields up to 10 g/L and has also been used successfully with DHFR-CHO cell lines. It is free from both royalty and license fees and is available to all holders of the GS Gene Expression SystemTM. Chemically defined and animal component free (CDACF) Developed by Lonza specifically for GS-CHO cell lines No royalties or license fees added Increased antibody yield Improved cellular metabolism Host Cell Protein Assays for GS-NS0 and GS-CHO Cell Lines Lonza has approximately twelve years of experience of developing host cell protein (HCP) assays for detection of host cell derived proteins from GS cell lines. In the Spring 2005 GS newsletter, we announced that the Western blot HCP assay had become available to our GS customers as a kit. In addition to this package, the GS HCP ELISA assay package is now available for use with GS-NS0 and CHOK1 cell lines with an assay for GS-CHOK1SV currently being developed for release at the end of 2008. The kits for the HCP ELISA (GS-NS0 and CHOK1 cell lines) and Western Blot (CHO, GS-CHO, GS-NS0 cell lines) are available for use by customers at their own site. Each kit contains reagents for five assays together with detailed protocols.The GS HCP Western blot assay package consists of: Anti-HCP antiserum: a key specialist component of the HCP assays. Raised against a cell extract prepared in Lonzas Development laboratories from a GS null (non-product expressing) cell line. The antiserum was specifically selected for use in a Western blot format that is used to detect and semi-quantify HCPs in recombinant products produced in GS-NS0 and CHOK1 cell lines. Specific HCP control: to ensure that the separation, electroblotting and probing procedures of the assay are acceptable. Know-how: a detailed methodology on how to perform the HCP Western blot. Expert technical support and service provided by the GS technical team.The GS HCP ELISA assay package consists of: Anti-HCP coating antiserum: purified coat antiserum prepared from the selected anti-HCP antiserum in the Western blot assay. The antiserum was specifically selected for use in the ELISA format that is used to detect and quantify HCPs in recombinant products produced in GS-NS0 and GS-CHO cell lines. Anti-HCP biotinylated antiserum: this is prepared from the purified coat antiserum. High and low HCP inter-assay control: to ensure that ELISAs performed on different days give comparable results. HCP assay standard: to produce a standard curve where the unknown sample HCP concentration can be extrapolated from the known relevant assay standard concentration. Know-how: a detailed methodology on how to perform the HCP ELISA. Expert technical support and service provided by the GS technical team. For customers for whom Lonza manufactures GMP products, we evaluate Lonzas generic assays to establish their suitability for testing the product cell line and the purified product. This evaluation is performed using the most suitable assay available to demonstrate the specificity and the accuracy of the HCP assays (Western blot is used to show the suitability of the antisera and ELISA). More recently, GS customers have requested Lonza to evaluate both formats of the HCP assays and this is now offered as a service. The evaluation service is performed at our Slough site and in the manner that we already use for our manufacturing customers. In brief, the purified product, the cell line supernatant and the cell medium are evaluated for specificity and accuracy in the appropriate HCP assays. By Western blot assay, we want to demonstrate that the major HCPs in the product cell line supernatant are detected by the anti-HCP antisera. In addition, we assess the cross-reaction with the anti-HCP antisera used in the assay. The HCP ELISA is evaluated to determine the minimum dilution at which the product can be tested and so retain the lowest limit of detection in the assay. In addition, the HCPs derived from the product cell line supernatant are assayed at different dilutions in the HCP ELISA to demonstrate parallelism and thus provide evidence of the similarity of the HCPs in the assay standard with those in the product cell line. After reporting the results of the evaluation to the customer, these generic HCP assays are available for the customer to use in-house. These assays can be used to detect HCPs in final product and in-process samples, and are suitable for the optimisation of purification processes.Services offered by Lonza HCP kitsKits for HCP ELISA (GS-NS0 and CHOK1 cell lines) and Western blot (CHO, GS-CHO, GS-NS0 cell lines) are available for use by a customer at their own site. Each kit contains reagents for five assays together with detailed protocols. On-site training and technical supportLonza staff can provide on-site (or at a Lonza site) training on the use of the HCP kits. HCP assay evaluation by LonzaLonza can evaluate your product for its suitability to be tested with Lonzas generic assays. The performance of the appropriate generic assay is evaluated; parameters include (but not exclusively) accuracy and specificity. HCP testing by LonzaLonza can perform HCP testing on samples provided by a customer. The product would first need to be evaluated for its suitability for testing using the generic assays. Sample testing would then be performed as and when required. HCP Product Specific Assay developmentShould the HCP assay evaluation show that the generic assays are not suitable for a particular product then a product specific assay can be developed by Lonza. Rapid Production of Antibodies by Pooled CHOK1SV Transfectants Cell line development is often a critical path activity that limits the availability of material for starting other studies, e.g. formulation buffer evaluation. The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programmes. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: In a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.Growth and productivity of GS-CHO pools in a disposable bioreactor Growth and productivity of GS-CHO pools in a disposable bioreactorThe pCon Plus Vectors The pCon Vectors are an easy way to re-express whole antibodies. The constant region vectors are a set of vectors offering a range of immunoglobulin constant region vectors cloned into the pEE vectors. These vectors offer easy construction of full length antibodies with human constant regions and the convenience of the GS System. Key Benefits Allows easy subcloning of variable regions - Single ligation step Fast construction of chimaeric or fully humanised antibodies Wide range of isotypes and allotypes available Gene optimized DNA sequences CONSTANT REGIONHeavy chain Light chain Isotype Allotype Isotype Allotype IgG1 f, za and zax Lambda2 N/A IgG2 n- Kappa Km(3) IgG3 g IgG4 N/A* *more stable hinge mutant availableNB: IgG1, IgG2 and IgG4 constant region genes are also available where the codon for the C-terminal lysine has been removedThe constant region vectors can be accessed under Research Evaluation Agreement with Lonza; email GSL for more details.What is the difference between using NS0 or CHO cells for preparation of antibodies? The glycosylation of these two cell lines is different. It is generally thought that the glycosylation patterns of proteins from CHO cells are closer to those in humans. However, as antibodies are not heavily glycosylated, the glycosylation pattern tends to be less of an issue than with other types of products. There have been a number of reports of NS0 glycosylation affecting function when compared to the same product expressed from CHO cells. If the glycosylation pattern of the antibody is known to be important to its function then a comparison of the product produced both in CHO and NS0 cells may be required. The antibody products prepared for clinical trials at Lonza are expressed in either NS0 or CHO cells. There are currently four licensed products that are produced using GS-NS0 cell lines. Relevant references are included below.Bebbington C.R. (1991): Expression of antibody genes in non-lymphoid mammalian cells. In Methods: A Companion to Methods in Enzymology 2: 136-145.-Lifely, M.R., Hale, C., Boyce, S., Keen, M.J. and Phillips, J. (1995). Glycosylation and biological activity of Campath - IH expressed in different cell lines and grown under different culture conditions. Glycobiology 5, 813-822.-Robinson, D.K., Chan, C.P., Yu Ip, C.C., Seamans, T.C., Lee, D.K., Lenny, A.B., Tung, J.-S., Di Stefano, D.J., Munshi, S., Gould, S.L., Tsai, P.K., Irwin, J., Mark, G.E. and Silberklang, M. (1994). Product consistency during long-term fed-batch culture. In Animal Cell Technology: Products of Today, Prospects for Tomorrow. Butterworths-Heinemann, pp763-767.-Robinson, D.K., Chan, C.P., Yu Ip, C., Tsai, P.K., Tung, J., Seamans, T.C., Lenny, A.B., Lee, D.K., Irwin, J. And Silberklang, M. (1994). Characterization of a Recombinant Antibody Produced in the Course of a High Yield Fed-Batch Proce