Pharmacopeial Monographs for Biotechnology Products

1. Pharmacopeial Monographs for Biotechnology Products Anthony Mire-Sluis Executive Director – Global Product Quality and External Affairs This presentation contains the personal opinions of the author and does not necessarily reflect a company wide position on the contents herein

2. Specific Considerations for Protein Products

3. There Are Special Considerations For The Development of Monographs for Biotechnology Products • Biotechnology products are different from classical small molecule drugs as they tend to be • More complex in structure • Heterogeneous • Produced by fermentation/bioreaction of living organisms • Generally have poorly understood structure/function/clinical efficacy relationships • Can induce immune responses with clinical impact

4. Biotechnology Products Are Naturally Complex • Biotechnology products never consist of a single moiety: • Product related substances • Protein variants that have similar biological activity to the ‘parent’ molecule • Product related impurities • Protein variants that do not have similar biological activity to the ‘parent’ molecule • Process related impurities • Host Cell Proteins • Endotoxins • Host cell DNA • Media components • Residual processing agents (e.g. protein A, process chemicals) • Process Contaminants • Adventitious agents • Leachables

5. Protein Product Characteristics Primary Structure: Trp-Val-Tyr-Ala-Cys-Arg-Trp-Glu-Ala-Cys-Phe-Thr-Arg-Asp Secondary Structure: Trp-Val-Tyr-Ala-Cys-Arg-Trp Asp-Arg-Thr-Phe-Cys-Ala-Glu Tertiary Structure: Post-translation Modification: Quaternary Structure: Heterogeneity:

6. Acetylation Acylation Addition of lipid Amidation/Deamidation Carbamylation Carboxylation Cleavage Formylation Gamma Carboxyglutamic acid O-linked Glycosylation N-linked Glycosylation Methylation Methionine Oxidation Norleucine Phosphorylation Sulphation Examples of Post Translational Modifications of Protein Products

7. Understanding Concerns with Protein Variants and Process Materials

8. The Concerns With Product and Process Related Materials – What are the Critical Quality Attributes relevant for a Monograph? • Clinical Relevance • Any known correlation of material to impact: • Pharmacokinetics • Bioavailability • Pharmacodynamics • Efficacy • Safety/toxicity • Immunogenicity • Genotoxicity • Carcinotoxicity • Stability • Will the product still work? • Are degradation products clinically relevant? • Consistency of Manufacture • Can you make the same product batch to batch?

9. Linking Product Characteristics to Clinical Parameters is Vital to Define Critical Quality Attributes and can be Manufacturer Specific Process Change Product Structure And Function Impurity Profile Adventitious Agents Quality Safety Efficacy

10. Examples of Variability/Heterogeneity that can Affect the Requirements of a Product Specific Monograph

11. Glycosylation • Potency can be affected if the molecule: • Has Fc function • Has glycosylation in the Fab region • Affect on PK highly dependent on sugar moieties • High mannose can clear quicker in vivo • Bioavailability depends on any charge differences • Not affected on stability • Not that easy to control during manufacture lot to lot

12. Fucose Galactose N-Glycan Types High Mannose (high-mannose) Hybrid Complex Mannose GlcNAc

13. Potential Biological Impact of Glycosylation Differences – High Mannose • Clearance of the protein: • Asialoglycoprotein Receptor (liver) • - Recognizes Gal and GalNAc • - Clustering increases Affinity • Mannose Receptor (liver) • - Recognizes Man, GlcNAc, Fuc • - Some indication of Clustering • Antigenicity: • Clearance through Mannose Receptor and presentation by APC • Safety • Complement Activation through Mannan Binding Protein

14. Using Non-Clinical Studies to Define the Criticality of Glycosylation of a MAb Product Fuc Gal - GlcNAc - Man | Man - GlcNAc - GlcNAc - Asn297 Gal - GlcNAc - Man • Variability due to: Terminal Galactose Core Fucose Oligomannose vs. Biantennary Non-glycosylated • Potential Impacts on: Complement-dependent cytotoxicity Antibody-dependent cytotoxicity Antigen binding Pharmacokinetics AAPS 2004

15. Relative Distribution of MAb Glycopeptide Forms Following In Vivo Exposure Other groups in study yielded similar results, oligosaccharide profile unchanged over time. ~70% of Xolair cleared after 4 days R. Keck et al 2004

16. Aggregation – Not all Aggregates are the Same Non Dissociable Dimer Non Dissociable Aggregate Dissociable Dimer

17. Aggregation is a Major Factor in Product Immunogenicity • Presence of aggregates precludes tolerance in naïve immune system • Tolerance to IFN-a broken by aggregates • Immune response to rIL-2 attributed to microaggregates: antibody development in 47-74% • MAb: elimination of aggregates eliminated immunogenicity • Enhancement of immunogenicity by other product changes (e.g. oxidation) may act through promoting aggregates

18. How Aggregation Affects the Immunogenicity Process Cytotoxic T-cells/NK cells Naïve T cell Activated T cell Helper T cell TCR Cytokines MHC Resting B cell APC Activated B cell Aggregated Therapeutic Protein Antibodies

19. Dimer That Dissociates on Dilution – Is There a Concern? Dissociable Dimer

20. Aggregation – Not All Aggregates are the Same – Dimers, Trimers versus Multimers Spacing: 60Å 85Å 140Å 190Å 100% TNF-a SA 50% 20% 10% 100% 0% + 101 102 103 104 105 106 GMT

21. Non Dissociable HMW Aggregates Non Dissociable Aggregate • Potency often reduced • Affects on PK not well documented but likely to be • different from monomer • Bioavailability likely to be affected • Can increase on stability • Should be able to control during manufacture lot to lot • Immunogenic potential high

22. Non Dissociable Dimer Non Dissociable Dimer • Potency can be reduced • Affects on PK not well documented but likely to be • different from monomer • Bioavailability likely to be affected • Can increase on stability • Should be able to control during manufacture lot to lot • Immunogenic potential appears less of a concern

23. Process Impurities can have Biological Consequences • Residual DNA not a concern if you can get it below 10ng/dose – the WHO recommended level • Immunogenicity is an issue for host cell proteins – including hypersensitivity reactions in addition to acting as adjuvants to induce immune responses against the protein product • Media components can also be an immunogenicity concern (e.g. yeast proteins) • Process chemicals can be a toxicity issue (residual solvents, small molecules etc.) • You have to get rid of viruses and microbes!

24. Specific Concerns on how Protein Product Monographs Would be Written to Assure Product Quality

25. Monographs are only as good as the Analytical Techniques they contain • By the time a product is licensed, a company will have developed highly product specific assays that take extensive time, money and often novelty – this can lead to Intellectual Property issues with biosimilars • As the lifecycle of a product proceeds, tests may be dropped as the innovator proves process consistency or through validation – how would one manage a new biosimilar? • New technologies to characterize biologicals are being developed all the time, so pharmacopeias should assure continuous improvement in biological monographs • Some assays (e.g. bioassays or immunoassays) may utilize product specific reagents – how to deal with that?

26. Setting Limits for Compendial Tests for Biological Products • For small molecules, the API is usually homogenous of high purity, very reproducible lot to lot, tests are generally highly precise and are able to detect most low level impurities • For proteins, manufacturing processes produce heterogeneous material with a certain amount of variability lot to lot, tests are generally more variable (e.g. gel based methods) and often not as sensitive • Does one use the innovators specifications as limits? • Specifications alone do not assure product safety/efficacy • May include terms such as ‘no new peaks within assay variability’ which requires a reference standard and understanding of assay precision • Specification limits can be altered over time during a product lifecycle – depends when a monograph is written (post patent expiry?)

27. Process Related Impurities are a Special Concern for Protein Products • The nature and quantity of process related impurities is highly manufacturer dependent, so how they are covered by a monograph is not simple • Specifications for process related impurities are often dropped prior to licensure or post licensure through process validation (e.g. residual protein A, HCPs, DNA etc). • Some process related impurities may be specific to a particular manufacturer (e.g. HCPs, process contact leachables, specific buffers etc.) • Others may be related to manufacturer specific container closures or formulations (e.g. vials versus prefilled syringes, different excipients etc.)

28. Process Related Impurities are a Special Concern for Protein Products – HCPs • Protein products contain host related proteins that are very specific to the cell line and process clearance mechanisms • It is not just the amount that is important, but the actual types of HCPs in the product – since they could be immunogenic in extremely small amounts • Only manufacture/product specific preclinical and clinical studies can assure safety • Therefore, simply requiring the level of HCPs to be met in a monograph is not appropriate • Propose to include ‘Host-cell-derived proteins : the limit is approved by the competent authority’ but not sure how one controls for the types of HCPs (not often revealed in specification testing)

29. Process Related Impurities are a Special Concern for Protein Products – Media/Culture Components • Media components can be manufacturer specific, thus it is difficult to prescribe any tests for residuals in the product • For example, soy hydrolysates and yeastolates can result in the presence of beta-glucans in the product • Beta glucans can cause immune reactions in patients and requires some level of monitoring • Other additives can be manufacturer specific (e.g. methotrexate, stimulators of protein secretion or types of anti-foam) • Tests are often ‘validated out’ by clearance studies, or having a specification, but this can be manufacturer specific depending on the process • Propose to include: ‘Process Related Impurities : the type and limits are approved by the competent authority’

30. Glycosylation Requirements are Difficult to set Appropriately • As described, glycosylation patterns are heterogeneous and can vary from batch to batch. • The acceptable variability is defined by preclinical and clinical studies whereby the role of glycosylation in PK, bioavailability, clearance and potency are established • The analytical tests to define glycosylation profiles go way beyond simple isoform patterns and would be difficult to include in a monograph – similar IEF patterns does not assure comparability • Existing monographs for glycosylated biotech products have been limited due to the separate naming of different products with different glycoforms (e.g. Erythropoietin Alpha, Beta, Gamma etc.) • How different can a glycoform pattern be before it is no longer similar?

31. Potency Assays • Bioassays often require company specific developed cell lines • Therefore should be general –‘Carry out the assay using a suitable method such as the following, which uses the conversion of a tetrazolium salt (MTS) as a staining method. Alternative methods of quantifying cell proliferation, such as measurement of intracellular ATP by luciferase bioluminescence, have also been found suitable, and may be used as the assay readout, subject to appropriate validation. The assay conditions (for example, cell concentration, incubation time and dilution steps) are then adapted accordingly.’ • What to do if an International Standard is not available? • Use of mass units is not meaningful without a reference standard being made available

32. The Requirement to Have a Reference Standard for Biological Products • Product specific monographs refer to a ‘reference standard’ for many of the tests they include • The provision of material for a standard at the quantity required can provide competitive advantage to biosimilars, especially if a monograph is produced during the in-patent lifetime of a product • Many biologicals are liquids that require validated storage, container closures and transport conditions – thus making a compendial standard harder to create for long term distribution worldwide • Can monographs be written that do not require a reference standard? • Inclusion of chromatograms, retention times, molecular weight markers and appropriate limits around numbers of variants

33. The Need to Assure Stability of Biological Products • Protein products tend to be much less stable than chemical drugs – they are most often liquid formulations and are kept at 2-8 degrees • The stability profile of biological products can not be predicted by lot release tests, yet there is no inclusion of tests that assure the appropriate degradation profile in current monographs • The formation of degradation products from biologicals can have clinical consequences (e.g. immunogenicity, decreased potency) and tests on fresh material do not assure two different products will degrade in the same way – thus they could look quite different as time progresses

34. The Concern over Interchangeability of Biotechnology Products • There is a great deal of industry concern that a monograph could give the ‘green light’ for the interchageability of biotechnology products • Unlike small molecule products, the analytical testing prescribed in any monograph/s for biotechnology products is highly unlikely to assure clinical comparability for the many reasons discussed • Some developing regulatory authorities regard compendial tests and limits as ‘the’ quality requirements to license a product

35. The Concern over Interchangeability of Biotechnology Products • Therefore, it is proposed to include:‘This monograph includes analytical methods and acceptance criteria that provide the minimum analytical product quality required to be compliant with the pharmacopeia. Due to the complexity of biotechnology manufacturing processes and the complex relationship of product structure, function and purity to safety and efficacy, analytical characterization alone can not assure safety and efficacy or substitutability of biotechnology products. Therefore, the tests contained within this monograph must be considered in addition to more extensive analytical characterization, preclinical studies and clinical studies to assure comparability of biotechnology products.'

36. Other Considerations for Discussion • Different categories of monographs – Drug substance and/or Drug product • Could be useful to distinguish different container/closures, final formulations, liquid versus lyophilized etc. of similar drug substance • The ‘flexible’ monograph could allow for differences in manufacture that only affect certain aspects of a drug substance quality profile • Defining critical product characteristics versus the GMP process specific (usually approved by regulatory authorities)

37. Conclusions • Developing appropriate, meaningful monographs for protein products requires considerations beyond those used for small molecule products • Selecting appropriate tests and limits becomes highly product dependent and needs to consider any potential differences in manufacturing processes • An understanding of Critical Quality Attributes is important for biological products in order to define an appropriate set of tests • Unlike small molecules, it is generally accepted that a set of analytical tests alone does not assure product safety or efficacy and some level of preclinical/clinical testing is required • Therefore, ensuring that monographs for biotechnology products are seen as the ‘necessary but not sufficient’ and that meeting those requirements does not assure interchangeability is essential