1. Industrial Cell Culture Features Related to Large Scale

2. 2 Features of Industrial Cell Growth Cleaning and sterility Types of culture control Batch-to-batch consistency

3. 3 Cleaning and Sterility Desire single cell type: host cell Avoid contamination/infection –Toxin risk –Purification problems –Overgrowth of host cells Avoid contamination during manipulations Sterilize components prior to use

4. 4 Media Filter Sterilization

5. 5 Clean in Place Tank is cleaned where it sits Connected to pump system –Caustic –Water –Acid –Water All surfaces contacted by liquids

6. 6 Sterilize in Place All lines into fermentor must be sterilizable Tank itself is steam- filled under pressure Clean steam used –Lines linked by valves –Valves are gas tight

7. 7 SIP Skid

8. 8 Types of Culture Control Larger cultures are harder to maintain Want highest possible cell density –Product yield increases –Overhead costs decline Closed system has low density limits –Continual decrease in nutrients –Continual increase in waste –Limited productivity

9. 9 cell number limiting nutrient product Closed System (batch culture) amount time

10. 10 Fed Batch Extends culture life, so cell number higher –May involve partial medium replacement –May involve addition of specific nutrients limiting nutrient amount time

11. 11 Continuous Culture Constantly remove and replace medium –Removal rate depends on cell growth rate –Removal allows constant density (not necessarily high) Perfusion vs. chemostat amount time cell number limiting nutrient product

12. 12 Simple Chemostat

13. 13 Batch-to-batch Consistency For FDA approval, process must be reproducible –Continuous cell lines can “drift” over time –Chromosomal abnormalities –Phenotypic changes Approval granted subject to “invariance” –Cells must be stable –Duration of production

14. 14 Master Cell Bank “Inexhaustible” supply of cells –All frozen at same stage –All product can come from “same” cells

15. 15 12345678 Making a Master Cell Bank Start with initial engineered cells and split as far as possible Each container becomes seed stock in working bank

16. 16 Working Cell Bank Made from one sample from master bank –Expand to create secondary supply stock –Number of units depends on planned use One batch drug per culture starter –Planning life-span of drug vital –Going back to master bank requires FDA adjustment

17. 17 Example Biogen uses CHO cells to make  - interferon Working bank consists of 1 mL vials –10 7 cells frozen per vial –One vial grown to 2000 L in culture train –Product from 2000 L is a batch –One month from vial to pre-formulation Two batches per month Working cell bank started with enough for ~ 42 year run

18. 18 Validation Issues Cells from actual bank to be used –Grow reproducibly –Produce reproducibly –Purification validated –“Process is under control” If disaster arose, new vial from master used –Revalidate a new working bank

19. 19 Review Scale makes process more difficult –Keeping aseptic conditions –Controlling growth of lots of cells –Maintaining consistent process Careful validation and documentation is key