1. Dock to Stock at Skyline Biotech A brief Outline of how we make our product at Skyline Biotech

2. What is a Tech expected to do? Focus on –Quality –Your training –The facilities around you –The Utilities that you use Operational Excellence

3. Dock to STock A process overview How exactly do we go from our raw materials to a finished product

4. Alpha Amylase From Bacillus subtilis

5. Training Goals Describe the main functions of the manufacturing process Explain how these processes come together to make a product

6. Master cell Bank Large volume of the original Strain or construct is held under cryogenic preservation. –Usually in 1ml ampoules –Usually in liquid Nitrogen Access is limited to ensure integrity of product.

7. Working Cell bank Created from the master cell bank for producing batches of product Do not need to continually go to Master cell bank A culture of the strain that makes your product –Continuously checked for efficiency and purity –When parameters are not met then another WCB is made from MCB

8. Raw Materials Media for the cells Quarantined and tested for quality before being used in manufacturing Many positions exist to ensure the consistency and quality of the Raw Materials chain A list of accepted suppliers is kept A list of accepted substitutes is kept

9. Process Paperwork Forms, Formulas and SOP are kept current and up to date Small changes may be made but must be proved to not change the process All completed paperwork is kept Typically people with experience in writing or the process are involved

10. Small Scale Cell culture A sample from the WCB is transferred to successively larger culture tubes Selective Media used to keep strain Check for –Cell density –pH –Temperature –Nutrient concentration Aa, sugar, oxygen, carbon dioxide

11. Continuously check for Quality Quantity consistency

12. Large cell Culture Typically a culture is not diluted more than 10x between each fermentation exchange Fermentation process is Batch –Cells are grown until they reach a certain density then transferred en mass to a new vessel with new media

13. How important is sterile technique?

14. Product Recovery Harvest. This depends on where the product is –In cells Separate the cells out, break open cells and start product purification –In media Separate the cells out, start product purification from the spent media

15. Types of Harvest TFF Tangential Flow Filtration Continuous Flow Centrifugation

16. Purification, Isolation and Viral inactivation TFF or column chromatography is used to separate proteins by size, charge, affinity or hydrophobic. The product will be separated by a complex mixture The product may not be absolutely pure –However if the process is the same the contaminants are validated with the final product

17. Viral Inactivation

18. Formulation and pool bulks Formulation –Concentrated protein is formulated to become tolerable to the patients –Ensures that the product is compatible in blood pH 7.1 –Bulk is frozen and stored in freeze/thaw tanks to await filling

19. Filling Final vials are filled under as sterile conditions as possible

20. Freeze drying Process increases shelf life and product stability

21. Product inspection Empty vials and stoppers are checked Filled are checked Also perform a container integrity test

22. Label Final Product

23. Final Product is packaged.

24. Alpha Amylase is ready to use