Current Practice in Bioanalytical Methods for Biosimilars:Case Study of Rituximab Assay Development for PK, ADA and Nab Xun Wang, Ph.D. Department of Translational Medicine QPS, LLC
Top 10 Prescription Drugs in 2016 (*) Drug Name Innovator Indication Mech. Of WW Sales Billion ($) (Patent Expiry) Company Action 2009 →2016 Humira™ (2016) Abbott Rhematoid Arthritis TNFαMAb 5.6 → 10.1 Avastin™ (2019) Roche Colorectal Cancer VEGF MAb 5.7 → 8.9 Enbrel ™ (2012) Pfizer, Amgen & Rhematoid Arthritis TNFα Inhibitor 6.5 → 7.3 Takeda Rituxan™ (2018) Roche Non-Hodgkins Anti-CD20 MAb 5.6 → 6.8 Lymphoma Crestor ™ (2012) AstraZeneca/ Anti-Hyperlipidemics Small Molecule 4.8 → 6.3 Shionogi/Chiesi Chemistry Herceptin™(2019) Roche Breast Cancer Her2/neu MAb 4.9 → 6.2 Remicade ™ (2014) JNJ Rhematoid Arthritis TNFα MAb 5.9 → 5.7 Lantus ™ (2018) Sanofi Aventis Diabetics Insulin 4.3 → 5.3 Advair ™ (2011) GSK Bronchodilators Small Molecule 8.0 → 5.2 Chemistry Prolia ™ (2013) Amgen/Daichii Osteoporosis Bone Calcium 0.0 → 5.2 Sankyo/GSK Reg. MAb (*) 8 out of the 10 top-selling drugs in the US are biologics. By 2016, some of which will face biosimilar entry.
BiosimilarThe biological product is highly similar to the reference productnotwithstanding minor differences in clinically inactive components,” andthat “there are no clinically meaningful differences between the biologicalproduct and the reference product in terms of the safety, purity, andpotency of the product.” Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(2) of the PHS Act.
Biosimilar Industry Today Market Intelligence $378M in July, 2010 – June, 2011 Forecast to rise to $2.6B by 2015 Development Cost $2M – $4M (small molecule generic) $100M - $200M (large molecule biosimilar) Key Players: Innovator companies, Emerging market Major Alliances announced in recent years Amgen -Watson: Oncology antibody products Roche - Emcure Pharmaceuticals (India CMO): Herceptin and Mabthera Merck - Parexel Biocon (India) - Mylan: Biosimilar monoclonal antibodies Pfizer - Biocon: Insulin and insulin analogs (The deal was prematurealy terminated in March) Momenta (Cambridge, MA) – Baxter International: Enoxparin plus 6 other unspecified biosimilars Biogen Idec – Samsung (Korea)
FDA Guidance Public Health Services Act (e.g., erythropoietin) The Biologics Price Competition and Innovation Act of 2009 (BPCI). Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product (February, 2012). Scientific Considerations in Demonstrating Biosimilarity to a Reference Product (February, 2012). Biosimilars: Questions and Answers Regarding Implementation of the Biologics Price Competition and Innovation Act of 2009 (February, 2012).
Bioanalytical Approaches PK Assay: 1 Assay vs 2 Assays Reference standard QCs ADA/Nab Assays: 1 Assays vs 2 Assays Positive controls Comparable activity and sensitivity
QPS Validated Bioanalytical Methods forBiosimilar Currently QPS has validated methods for the following biosimilars of market potential: Anti-Factor IIa and Anti-Factor Xa assays for Enoxaparin (Activity Assay) Anti-Factor IIa and Anti-Factor Xa assays for Dalteparin (Activity Assay) Rituximab (MabtheraTM, RituxanTM): Human PK method (ELISA, Gyrolab) Rituximab Human ADA methods (MSD ECL) Rituximab Human Nab methods (Cell based functional) Bevacizumab (AvastinTM) Human PK method (ELISA) Darbepoietin Human PK method (ELISA) Trastuzumab (HerceptinTM) Human PK method (ELISA) Cetuximab (ErbituxTM) Human PK method (ELISA) FSH (PuregonTM, Gonal-FTM) (Immulite)
Case Study: Rituximab PK, ADA and Nab Rituximab (Mabthera, Rituxan) is genetically modified monoclonal antibody that targets CD20 on B cell surface. Treat solid tumor of lymphoid cells and rheumatoid arthritis with excessive or dysfunctional B cells. The mechanisms of action is not fully understood. Direct signaling Complement-dependent cytotoxicity Antibody-directed cell-mediated cytotoxicity
Case Study: Rituximab PK PK Assay Development (ELISA) 2 1 Assay 1.5 1 Reference standard: Innovator 0.5 QCs: Innovator and Biosimilar 0 10 100 1000 10000 Rituximab Concentration (ng/mL) y = ( (A - D)/(1 + (x/C)^B ) ) + D: A B C D R^2 Standard Curve (Std: Conc. vs OD) 0.064 1.22 3542.079 3.361 0.999
Case Study: Rituximab PK PK Assay Development (ELISA)* Validation Items Results Assay Range 100 - 5000 ng/mL (HUSE) QC intra/Inter Precision 2.9% to 10.7% QC intra/Inter Accuracy -8.8% to 9.5% Matrix Selectivity (low and high QC) At least 85% within ±25% for solid tumor and RA serum lots Dilution Linearity 500,000 ng/mL diluted up to 2,000–fold; ADA Interference At least 1 µg/mL at low QC and 10 µg/mL at high QC Stability in HUSE -200C/-700C/RT/FT * Data shown here are from the Rituximab Innovator
Case Study: Rituximab PK PK Assay Development (Gyrolab) Alexa labeled - anti human IgG A high throughput assay platform with Ab miniaturization and automation Rituximab Flow Biotinylated-Rat Wide assay range: 90 – 60,000 ng/mL in HUSE anti ID rituximab Validated for Innovator SA analysis Streptavidin-coated bead X. Liu et. al. Journal of Immunological Method, 2012
Case Study: Rituximab ADA ADA Assay Development 2 Assays (Innovator and Biosimilar) Assay format: Bridging with labeled Innovator or Biosimilar Positive controls • Anti-Rituximab Id • Rabbit anti-Rituximab Polyclonal • Anti-Biosimilar Id
Case Study: Rituximab ADA ADA Assay Development (Innovator)* Validation Approaches Results Items Screening cut point At least 50 individual ST or 1. Floating cut point (SCC) RA lots 2. Significantly different SCPs between matrixes 3. Similar between Innovator and BSI Confirmatory cut At least 28 individual ST or Similar CCPs between innovator and point (CCP) RA lots spiked with drug BSI PC Crossing Check Anti-Biosimilar/Innovator PC showed similar activity in both Abs in Innovator/BSI assay innovator and BSI assays Assay sensitivity Determined for all three Sensitivities are similar between positive control antibodies innovator and BSI assays (< 20 ng/mL) Drug Tolerance Determined at low and high Low and high PC can tolerate at least PC (50, 400 ng/mL) 12 and 50 µg/mL Rituximab. * Data shown here are from the Rituximab Innovator
Case Study: Rituximab Nab Nab Assay Development 2 Assays (Innovator and Biosimilar) Assay format: Cell based • Binding? • Functional -- Go Positive controls • Anti-Rituximab Id • Rabbit anti-Rituximab Polyclonal • Anti-Biosimilar Id
Case Study: Rituximab Nab Nab Assay Development Rituximab MOA: • Complement-dependent cytotoxicity (CDC) • Antibody-dependent cell-mediated cytotoxicity (ADCC) • Apoptosis • A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody (Gazzano-Santoro et. al., Journal of Immunological Methods, 1997) http://minimednews.wordpress.com/
Case Study: Rituximab Nab Nab Assay Development Method Development Cells growth and density curve: WIL2-S Rituximab dose killing curve: 70-80% killing Rabbit complement dose curve Incubation time Matrix Evaluation (ST and RA): 1:100 • Specificity • Selectivity Compare the biosimilar to the innovator PC cross check
Case Study: Rituximab Nab Nab Assay Development (Innovator)* Validation Approaches Results Items Screening cut point At least 50 individual ST or 1. Floating cut point (SCC) RA lots 2. Significantly different SCPs between matrixes 3. Similar between Innovator and BSI Immunodepletion At least 28 individual serum Similar between innovator and BSI lots treated with Protein A/G PC Crossing Check Anti-Biosimilar/innovator PC showed similar activity in both Abs in Innovator/BSI assay innovator and BSI assays Assay sensitivity Determined for all three Currently ongoing (~ 50 µg/mL) positive control antibodies Drug Tolerance Determined by High PC Can tolerate 10 µg/mL Rituximab at titering in the presence of the estimated assay sensitivity various drug levels. * Data shown here are from the Rituximab Innovator
Conclusion PK Assay: One assay is sufficient to measure both innovator and biosimilar drugs. • Reference Standard: Innovator/BSI • QCs: Innovator/BSI Immunogenicity (ADA and Nab) requires two assays, which are best to develop simultaneously and achieve: Similar screening and confirmatory cut points Similar sensitivity Similar PC cross reactivity Similar drug tolerance
Acknowledgement Joy He Avery Tolosa Jiannian Zhou Frank Liu YinLing Li Margaret Ma Laurelle Calliste Breann Barker Christina Xia Roni Weaver Yun Shen LingSing Chen Hui Zhang Holly Shen Chad Asher