Industry View on the Relative Importance of Clonality of Biopharmaceutical-Producing Cell Lines

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1 Industry View on the Relative Importance of Clonality of Biopharmaceutical-Producing Cell Lines Christopher Frye, Eli Lilly & Company, Indianapolis IN Rohini Deshpande, Amgen Inc, Thousand Oaks CA Scott Estes, Biogen Inc, Cambridge MA Kathy Francissen, Genentech Inc, South San Francisco CA John Joly, Genentech Inc, South San Francisco CA Anthony Lubiniecki, Janssen R&D LLC, Malvern PA Trent Munro, Amgen Inc, Thousand Oaks CA Reb Russell, Bristol Myers Squibb, Pennington NJ Tongtong Wang, Eli Lilly & Company, Indianapolis IN Karin Anderson, Pfizer Inc, Andover MA

2 Introduction What is clonality? Why are production cells cloned? Can clonality ever be achieved in an absolute sense? What are the major Health Authority regulatory requirements? What is the role of clonality in the evaluation of product quality?

3 What is Clonality? Dictionary Definitions ability to be cloned (Dorland, Miller-Keane) ability to form clones (Saunders Veterinary) state of proliferation determined by the cell(s) of origin (McGraw-Hill Concise) For our purposes, we suggest that none of these are suitable, and what we probably mean by clonality is the degree of certainty that a cell line represents the progeny of a cloned cell

4 Why Are Production Cells Cloned? Production cells are cloned in order to Identify cells with high production levels Identify cells which make product of appropriate quality and post-translational modifications Identify cells adapted to thrive under production conditions, e.g., Possess adequate growth rate Maintain high viability for extended time periods Limit the pre-existing genetic variation of the cell line reflecting prior random mutations to be represented in the production cell line

5 Limiting Pre-existing Random Variation PDL 1 PDL 2 PDL 3 PDL 4

6 Can Clonality Ever Be Achieved in an Absolute Sense? It is possible to subject a mixed cell population to a cloning procedure which has a high probability of generating multiple separate populations of cells, many of which will likely have descended from a single cell. These are typically termed clones. The level of proof for each clone having been derived from a single cell varies with the method & technology employed, and is a probabalistic estimate rather than an absolute fact.

7 Can Clonality Ever Be Achieved in an Absolute Sense? It is impossible to use cloning to create a culture in which all cells are genetically identical due to the inherent minimum mutation rate of DNA replication, even under ideal circumstances unlikely to exist in immortalized cell lines. By the time that enough cells could be grown from a single cell to create a new cloned culture, further random DNA mutation will have occurred after cloning to ensure that some of the progeny cells are not identical to the single parental cell. This is an inherent property of DNA replication in all known life forms. Selective pressure agents & use of immortalized cell lines may further increase the rate of further random DNA mutation after cloning has occurred.

8 Can Clonality Ever Be Achieved in an Absolute Sense? So it is possible to achieve the act of cloning, and to be reasonably (but not totally) sure that the resulting culture is derived from a single cell, but it is not possible to assume or conclude that all cells in the resulting culture are genetically identical.

9 What Are the Regulatory Requirements of Major Health Authorities Regarding Clonality? FDA EMA ICH Other

10 What Is the Role of Clonality in the Evaluation of Product Quality? Assuring product quality depends on developing an integrated control strategy based on a multifaceted evaluation of: Cell line history Genetic manipulations introduced into the cell line Process of cell identification and cell cloning Process & analytical development Process validation and analytical validation Raw material controls Product testing and characterization Comparability assessment when appropriate Evaluation of DS & DP stability Characterization of degradants Adequate understanding of mechanism of action

11 What Is the Role of Clonality in the Evaluation of Product Quality? Recognizing the intent of the regulatory guidelines and the value of a cloning step, nevertheless, it should be reiterated that incorporation of a cloning step cannot prevent genetic and phenotypic heterogeneity within the population of cells that arise post-cloning as these events are inherent to the use of immortalized mammalian cell lines. Thus, assurance of product quality and consistency must rely on the other tools described above.

12 Conclusions Clonality is an essential but inherently limited feature of any integrated control strategy for a modern biopharmaceutical product, and should be evaluated appropriately in order to provide adequate assurance of product quality, along with all of the other components of the control strategy. Collectively, these integrated control principles have worked well to provide a steady stream of over 100 therapeutic biopharmaceuticals which have significantly benefited public health, while building an admirable record from the standpoint of product consistency and safety over the past 30+ years. A version of this talk has been submitted for publication in the IABS journal Biologicals