Technical Challenges in the Development of Biosimilars. E. Morrey Atkinson, PhD Interphex May 1, 2012

Transcription

1 Technical Challenges in the Development of Biosimilars E. Morrey Atkinson, PhD Interphex May 1, 2012

2 FDA Guidance on Biosimilarity Guidance for Industry: Scientific Consideration in Demonstrating Biosimilarity to a Reference Product. Draft Guidance. U.S. Department of Health and Human Services, FDA, CDER, CBER. Feb Comparability of Biotechnological/Biological Products: ICH Topic Q5E. European Medicines Agency. June Guideline on Similar Biological Medicinal Products: European Medicines Agency. Oct The European Medicines Agency Approved the first Biosimilar in Current Biosimilars on the European Market include: Human Growth Hormones Human erythropoietins, Granulocyte-Colony Stimulating Factor

3 Need for Biosimilars Biologics Price Competition and Innovation Act of 2009 (BPCI Act) Created to allow abbreviated approval pathway for biological products which demonstrate they are highly similar to an approved biologic product. Saves time and resources not necessary to duplicate some human and animal testing.

4 Market Share of Biopharmaceuticals Biopharmaceuticals constitutes a large portion of the pharmaceutical market. Many of those patented biopharmaceuticals are coming off of patent. This brings the opportunity for consumers/government to save money. How much money are we spending on biopharmaceuticals? How much money do we save with generics? ~ 80% savings How much money can we save with biosimilars? Est. ~ 15-30% savings Why? Bioequivalence vs. Phase I and III clinical trial data, Process Costs Simeons, ClinicoEconomics and Outcomes Research 2011:

5 Impurity Definition Impurity An impurity is any component present in the excipient, drug substance, or drug product that is not the desired product, a product-related substance, or excipient, including buffer components. It may be either process- or product-related. GLOSSARY: USP Guideline

6 Defining Impurity Process-related impurities - As applied to the biologics and biotechnology areas, these are impurities derived from the manufacturing process. They may be derived from source tissue or host cells (e.g., protein, DNA), cell culture (e.g., inducers, antibiotics, or media components), or downstream processing (e.g., processing reagents or column leachables). Product-related impurities - As applied to the biologics and biotechnology areas, these are molecular variants of the desired product (e.g., precursors, certain degradation products arising during manufacture and/or storage), which do not have properties comparable to those of the desired product with respect to activity, efficacy, and safety. Product-related substances - As applied to the biologics and biotechnology areas, these are molecular variants of the desired product formed during manufacture and/or storage, which are active and have no deleterious effect on the safety and efficacy of the drug substance/drug product. These variants possess properties comparable to the desired product and are not considered impurities. GLOSSARY: USP Guideline

7 Quality Attributes of Biopharmaceuticals A-Mab: a Case Study in Bioprocess Development. CMC Biotech Working Group. Oct

8 Overall Differences and Similarities Similar: General Consideration for a Biopharmaceutical product Differences: Burden of Proof High Similarity Abbreviated Pre-Clinical and Clinical Trials No need for Dosage-Finding BLA vs. 351 (k) No 12 yr data exclusivity

9 Proof of High Similarity High Similarity between the Proposed product vs. Reference product Biosimilarity: Structure Function (Mechanism of Action) Non-Clinical Trials - Animal Toxicology Clinical Trials - Human pharmacokinetics, pharmacodynamics, immunogenicity, safety, efficacy * Drug Packaging may vary if shown that there is no effect on product: (i.e. delivery device, container closure, vial) * Sponsors are urged to discuss development program with FDA prior to clinical trials. Final decision on 351 (k) application.

10 Studies to Prove High Similarity High Similarity between the Proposed product vs. Reference product Biosimilarity: Structure Function (Mechanism of Action) Non-Clinical Trials - Animal Toxicology Analytical Studies on Drug Substance and Drug Product Animal Studies with Drug Product Clinical Trials - Human pharmacokinetics, pharmacodynamics, immunogenicity, safety, efficacy Clinical Study/Studies with Drug Product * Drug Packaging may vary if shown that there is no effect on product: (i.e. delivery device, container closure, vial) * Sponsors are urged to discuss development program with FDA prior to clinical trials. Final decision on 351 (k) application.

11 The Focus for Process Development Scientific: Proposed product vs. Reference product Biosimilarity: Structure Function Analytical Studies on Drug Substance and Drug Product Structure and Function affect the protein s safety, purity, potency, efficacy. Additional considerations: Excipients and product- and process-related impurities.

12 Protein Structure Three general ways that protein structure can be affected: 1.Primary amino acid sequence 2.Secondary, Tertiary and Quaternary structure (protein folding, protein-protein interactions) 3.Post-translational modification to amino acids (glycosylation, phosphorylation) Figure 6-21a Molecular Biology of the Cell ( Garland Science 2008)

13 Small vs. Large Molecules Insulin Amoxicillin

14 Major Size Differences within Biologics Antibody Insulin

15 Basic Immunoglobulin Structure Disulfide Linkages Light Chains Fab Regions Bind target antigen Independent binding regions (CDRs) Heavy Chains Fc N-Linked Glycosylation Site Fc Region Binds Fc Receptors on immune cells Effector Function mediates cell killing Receptor binding is dependent on glycoform structure (Fuc)

16 Protein Structure Wikipedia Raju. BioProcess Technical. April

17 Some Impurities and Related Substances Standard IgG Pyroglutamate Formation pyroe Chemical and Enzymatic Cleavage Disulfide Reduction or Scrambling Deamidation Glycoform Heterogeneity (K) (K) Met Oxidation C-terminal Lysine Variation

18 Protein Structure Scientific Considerations: Proposed product vs. Reference product Biosimilarity: Structure Function Analytical Studies on Drug Substance and Drug Product Three general ways that protein structure can be affected: 1. Primary amino acid sequence 2. Secondary, Tertiary and Quaternary structure (protein folding, protein-protein interactions) 3. Post-translational modification to amino acids (glycosylation, phosphorylation)

19 Introducing changes that affect Protein Structure STRUCTURE: Three general ways that protein structure can be affected: 1. Primary amino acid sequence 2. Secondary, Tertiary and Quaternary structure (protein folding, protein-protein interactions) 3. Post-translational modification to amino acids (glycosylation, phosphorylation) Where can these be introduced? Cell Line Upstream Process Downstream Process Formulation Packaging

20 FDA Guidance on Biosimilarity STRUCTURE: Orthogonal methods to draw conclusions - totality of evidence Multiple methods to draw conclusions about structure and function biosimilarity Qualitative and Quantitative methods Test multiple lots of proposed product Can include animal and human studies of PD and PK, along with human immunogenicity, safety and efficacy studies to totality of evidence for structure and function biosimilarity Drug Substance, Drug Product, excipients and impurities should be analyzed. MODERN TECHNIQUES

21 FDA Guidance on Biosimilarity STRUCTURE: Biosimilarity may be demonstrated even when there are minor formulation or structural differences i.e. post-translation modifications, Human Serum Albumin vs. Bovine Serum Albumin Demonstrate no effect on safety, purity, and potency of product.

22 FDA Guidance on Biosimilarity STRUCTURE: 1.Primary amino acid sequence Identical AA sequence *exception N- or C- terminal truncations that do not affect safety and effectiveness.

23 FDA Guidance on Biosimilarity STRUCTURE: 2. Secondary, Tertiary and Quaternary structure (protein folding, protein-protein interactions) Secondary, tertiary, quaternary structure Aggregate formation

24 FDA Guidance on Biosimilarity STRUCTURE: 3. Protein modifications to amino acids (glycosylation, phosphorylation) Examine protein modifications qualitatively and quantitatively. Protein variants (i.e. deamidation and oxidation) Intentional modifications (i.e. PEGylation sites )

25 FDA Guidance on Biosimilarity STRUCTURE: Amino Acid Sequence: Deduces from gene sequence and compared with analytical techniques. Peptide Map: Analysis of Selectively Fragmented Product by Mass Spectrometry, N-terminal Sequencing and Compositional analysis. Amino Acid Composition: Protein digestion analyzed by HPLC Terminal Amino Acid Sequence: Lysine at C-terminus of monoclonal Ab Sulfhydryl group and Disulfide bridges: Mass Spectrometry, Peptide Mapping (Reduced and Non-Reduced conditions) Glycan Analysis: Fluorescent labeling analyzed by Mass Spectrometry or HPLC

26 FDA Guidance on Biosimilarity STRUCTURE: Molecular Weight: Size Exclusion Chromatography, SDS-polyacrylamide gel electrophoresis, mass spectrometry Isoform Pattern: Isoelectric Focusing Extinction Coefficient: Determine based from UV/ Vis Spectrophotometry compared to a known concentration of product (Amino Acid Compositional Analysis or Nitrogen Determination)

27 FDA Guidance on Biosimilarity STRUCTURE: Electrophoretic patterns: SDS polyacrylamide Gel electrophoresis, isoelectric focusing, Western Blot, capillary electrophoresis Liquid Chromatographic patterns: Size Exclusion Chromatography, Reverse-Phase liquid chromatography, Ion Exchange Liquid Chromatography, Affinity Chromatrography Spectroscopic Profiles for Higher Ordered Structure: Circular Dichroism, Nuclear Magnetic Resonance Aggregation: Analytical ultracentrifugation, gel electrophoresis, High Presssure Size Exclusion Chromatography

28 FDA Guidance on Biosimilarity FUNCTION: Determine biological activity and potency using bioassays, biological assay, binding assays, enzyme kinetics Demonstrate the Mechanism of Action is similar with the same concentration of substance/product.

29 FDA Guidance on Biosimilarity Function Example: Show similar results with the same amount of reference and proposed product. Etanercept as example: Fusion protein of Tumor Necrosis Factor Receptor (TNFR) and Fc region (blocks TNF binding to TNFR) Bioassay: Cell based experiment. Spike in TNF in the presence and absence of Ref. and Proposed Product. Analyze the response on TNFR signaling. Biological Assay: Mouse model of rheumatoid arthritis treated in the presence and absence of Ref. and Proposed Product. Analyze the inflammation response in mice. Binding Assay: Analysis of the affinity of Ref. and Proposed product in binding to TNF.

30 Antibody Manufacturing Cell Culture Bioreactor Detergent Inactivation Low ph Virus Inactivation Clarification- Depth Filtration Polishing Chromatography Bulk API Presentation - Freeze/Storage Remove Virus Nano-filtration Centrifugation Membrane Filtration Protein A Capture Chromatography Purification Chromatography Concentrate/ Buffer Exchange TFF

31 Acceptable changes in quality attributes of glycosylated biopharmaceuticals Schiestl et. al. Nature Biotechnology. April (4) Purpose: Understand acceptable variation in biopharmaceuticals; hope to apply to biosimilars Approach: Analyse multiple batches of 3 biopharmaceutical products on the market from : Rituximab- mab Etanercept- TNFR/Fc fusion protein Darbepoetin alfa- modified form of erythropoietin

32 Acceptable changes in quality attributes of glycosylated biopharmaceuticals Capillary zone electrophoresis of Darbepoetin alfa : separates isoforms with different charges resulting from varying levels of sialic acid Shift in peak size by as much as 10% Post change: Increased Sialyation rate Schiestl et. al. Nature Biotechnology. April (4)

33 Acceptable changes in quality attributes of glycosylated biopharmaceuticals Glycan Profiling of Rituximab: Post change: Abundance of unfucosylated product increased by 3-fold. Schiestl et. al. Nature Biotechnology. April (4)

34 Acceptable changes in quality attributes of glycosylated biopharmaceuticals Lack of sialyation or fucosylation strenghtens the ADCC response. ADCC potency of Rituximab- Cell based assay Post change: ADCC response increased *Not as great of change on ADCC response as expected, perhaps structure plays a role. Schiestl et. al. Nature Biotechnology. April (4)

35 Acceptable changes in quality attributes of glycosylated biopharmaceuticals Glycan Mapping of Enbrel: Post change: N-glycan G2F decreased by almost 2 fold Schiestl et. al. Nature Biotechnology. April (4)

36 Acceptable changes in quality attributes of glycosylated biopharmaceuticals Conclusions: Manufacturers of Biopharmaceuticals are making process changes that affect the glycan profile of molecule and other quality attributes. This implies that variation is acceptable, even during a product s lifecycle. The challenge will be to determine the acceptable differences between biosimilars and the reference products. The FDA will examine the totality of evidence in making this determination.

37 Acknowledgements Angela Groehler, Ph.D.* Shumin Yang, Ph.D.