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Process development & production of glycosylated and sialylated follicle-stimulating hormone (fsh) in 'humanised' yeast.

Human recombinant fsh will be expressed in engineered yeast cells and purified to homogeneity to meet fsh quality currently marketed. The unique feature of the project is a cost-effective process of human glycosylation and sialylation of fsh, driven by human genes, in a non-human organism.

- Define Freedom To Operate: Decide on development strategy; Consult with patent attorney about the freedom to operate (PPS-PROTEIN PRODUCTION SERVICES LTD and GLYCODE). - Cloning and Expression: synthesis of different nucleotidic sequences to optimise secretion (test of some secretion factors and signal sequences). Cloning under inductible promoter (yeast expression vector). Transformation of N-glycosylation modified strains to express different glycoforms of rFSH. Expression in culture flasks and bioreactors to obtain product clones (GLYCODE). - Cell bank preparation and testing: Establishment of Master Cell Bank (MCB) from suitable clones according to criteria such as productivity, genetic stability, clone purity. MCB will be prepared in clean rooms under cGMP (Compliance (with) Good Manufacturing Practice) conditions (PPS). Safety testing to be contracted out to specialising CRO (Clinical Research Organisation). - Analytical methods development: Analytical methods covering in-process control and quality evaluation methods will be set up. These methods will include protein content, IEF, immuno-IEF, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) (Coomassie and silver stain), Western blot, RP-HPLC (Rapid Prototyping High Performance Liquid Chromatography), Size Exclusion Chromatography, peptide mapping, sugar content, glycosylation and sialylation pattern, ELISA (Enzyme-Linked Immuno-Sorbent Assay), Endotoxin and bioassay (GLYCODE and PPS). Characterisation methods e.g. CD, MS will be outsourced. - Bench scale evaluation: Selected clone expressing FSH as determined by ELISA assay will be cultivated in 100-500 ml flasks, induced to express the product for a predetermined time period. As the product is expected to be excreted to the medium, harvested supernatants will be used to quantify accumulated FSH by ELISA (GLYCODE and PPS). - Fermentation: The cultivation process will be scaled up and a selected clone will be grown in a suitable medium in a 5-20L fermentation vessel. Growth and expression conditions will be optimised (PPS). - Stability of FSH in harvest fluid: FSH stability in supernatant will be tested by storage at 4-8 C over a period of 14 to 30 days. Stability-indicating analytical methods will include ELISA and immuno-IEF (PPS). - FSH purification: Initial purification steps will include resin chromatography methods as reported in the scientific literature. Improvement and optimisation of the process will follow thereafter (GLYCODE and PPS). - Downstream process scale up: Purification process will be scaled up to production scale (PPS). - Bulk product stability: Stability of purified FSH will be tested at 2-3 temperatures for a period of three months at one month intervals. Stability-indicating analytical methods will include HPLC profile and IEF (PPS). - Additional analytical methods development and qualification: Establishment and development of analytical methods required for in-process testing and release of API (Active Pharmaceutical Ingredient) will be completed. The methods that are used for currently marketed products will be used as gold standard and reference. Methods to be used for product release will be qualified. Research Reference Standard (RRS) will be prepared from API (PPS). - Pre-clinical efficacy studies: Plan will be defined with specialising CRO (animal model study). Studies will be conducted using a commercial product as a reference (GLYCODE and PPS). - Pre-IND meeting: Pre-IND file will be prepared, including pre-clinical data, production process data and plans for production, toxicology, safety and clinical studies; Meeting with EMEA/CPMP will take place, and plans will be adjusted according to feedback (GLYCODE and PPS). - Engineering run; preparation of material for toxicology studies: Engineering run (fermentor) will be conducted and purification process up to API batch will be performed, using the same methods and equipment to be used in cGMP production. Formulated material suitable for toxicology study design will be prepared (PPS). - Toxicology and safety studies: To be out-sourced to specialising CROs. - Preparations for cGMP production: Master Batch Record for cGMP production will be prepared. SOPs and batch records for production and testing methods will be put in place; Facility and equipment will be validated (IQ/OQ); Cleaning of fermentors, columns, UF (Ultra Filtration) devices and process tanks will be validated (PPS). - GMP production and release of API for use: USP production; DSP production (API); Release testing of API; Characterisation testing of API (PPS). Provision of API material released for use in clinical trials; Preparation of PRB (Production Reference Batch) - to replace RRS. (PPS). - Stability testing of API: Stability-indicating methods will be defined. Stability of the material intended for use in clinical trials will be tested. - Documentation package: Development and qualification reports of process and analytical methods; Cell bank reports; SOPs; Pre-IND documentation; Production report and CoA of batches for use in clinical trials, Pre-clinical and tox studies; Stability report; IND document.
Acronym: 
FSH
Project ID: 
5 038
Start date: 
01-10-2009
Project Duration: 
36months
Project costs: 
1 640 000.00€
Technological Area: 
Biochemistry / Biophysics
Market Area: 
Other medical/health related (not elsewhere classified)

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