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Host Cell Protein Assays for GS-NS0 and GS-CHO Cell Lines
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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 Lonza’s 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 Lonza’s 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 kits
Kits 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 support
Lonza staff can provide on-site (or at a Lonza site) training on the use of the HCP kits.
• HCP assay evaluation by Lonza
Lonza can evaluate your product for its suitability to be tested with Lonza’s generic assays. The performance of the appropriate generic assay is evaluated; parameters include (but not exclusively) accuracy and specificity.
• HCP testing by Lonza
Lonza 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 development
Should 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.
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Rapid Production of Antibodies by Pooled CHOK1SV Transfectants
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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 |
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| The Latest GS Poster |
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The 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
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CONSTANT REGION
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Heavy chain
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Light chain
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Isotype
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Allotype
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Isotype
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Allotype
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IgG1
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f, za and zax
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Lambda2
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N/A
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IgG2
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n-
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Kappa
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Km(3)
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IgG3
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g
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IgG4
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N/A*
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*more stable hinge mutant available
NB: IgG1, IgG2 and IgG4 constant region genes are also available where the codon for the C-terminal lysine has been removed
The constant region vectors can be accessed under Research Evaluation Agreement with Lonza; email GSLonza@lonza.com for more details. |
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