MONOCLONAL ANTIBODY DEVELOPMENT AND PRODUCTION: TECHNOLOGICAL PRINCIPLES

MONOCLONAL ANTIBODY DEVELOPMENT AND PRODUCTION: TECHNOLOGICAL PRINCIPLES Antonio Moreira University of Maryland Baltimore County IV SYMPOSIUM SINDUSFARMA-IPS/FIP-ANVISA New frontiers in manufacturing technology, regulatory sciences and pharmaceutical quality system Brasilia June 22, 2015

Presentation Outline Characteristics of monoclonal antibody molecules Production technologies Quality control Recent advances Conclusions 2

Biopharmaceuticals on the Market Examples Interferons (Intron A, Pegintron, Betaseron, Avonex) Interleukins (IL2, IL11) Fusion Proteins (Enbrel, Orencia) Bexxar (Fully Murine) Remicade, Rituxan, Reopro, Simulect, Erbitux (chimeric) Herceptin, Avastin, Tysabri, Synagis, Zenapax (humanized) Humira (Fully human) Biosimilars (Remsima by Celltrion and Inflectra by Hospira) Antibody-Drug Conjugates Adcertis by Takeda Kadcyla by Genentech 3

Coordinates from Anatomy of the Antibody Molecule by E. Padlan, Mol.Immunol., 31, 169, 1994. PDB file 21g2 ; Picture made using RasMol: R. Sayle, E.J. Milner-White, TIBS, 20, 374, 1995

A Representative Antibody Molecule V L L Chain hypervariable regions C L V H S-S S-S H Chain hypervariable regions C H 1 S-S S-S Hinge region Intrachain disulfide bonds C H 2 C H 3 Carbohydrate 5

Current and future issues in the manufacturing and development of monoclonal antibodies. S. Kozlowski and P. Swann. Advanced Drug Delivery Reviews 28 (2006), 707-72, 707-722. 6

Complex Carbohydrate in Antibody Structure 7

Figure 2: List of FDA-approved antibody therapeutics. BioPharm International, February 2013 8

KEY TARGETS Developers are trying to create functional replicas of leading biologic drugs.sources: Company data, Biotechnology Information Institute Thayer, C&EN Houston. cen.acs.org. October 7, 2013. 9

Herceptin Binding to HER2 Receptor 10

Perjeta Mechanism of Action Step #4 PERJETA + Herceptin provide a more comprehensive blockade of HER2-driven signaling pathways. 11

Avastin ( Bevacizumab) Tumors release the VEGF protein causing nearby cells to sprout new vessels by angiogenesis. New blood cells feed the tumor Bevacizumab is an angiogenesis inhibitor Mechanism of action is by inhibiting vascular endothelial growth factor ( VEGF) Initially approved for colon cancer in 2004 Has been approved for us in other cancers such as: lung, renal, ovarian, glioblastoma 12

Trends in Upstream and Downstream Process Development for Antibody Manufacturing. P. Gronemeyer, R. Ditz and J. Strube. Bioengineering, 2014, 1, 188-212; doi: 10.3390/bioengineering1040188. 14

Recent advances in large-scale production of monoclonal antibodies and related proteins. A. A. Shukla and J. Thömmes. Trends in Biotechnology, Vol. 28, No. 5, 253-261. March 2010. 15

Bioreactor Operating Modes Batch Fed-Batch Continuous Perfusion 16

Table 1: Selected Perfusion Operations Issues: PRODUCT IL-12/23 Mab (Stelara)** TNF Mab (Simponi) Glucosidase alfa (Myozyme)** Galactosidase alfa (Fabrazyme)** Protein C (Xigris) Factor VIII (Kogenate-FS)** Interferon beta (Rebif)** IL-2 receptor Mab (Simulect) TNF mab (Remicade)** FSH (Gonal-F) Galactosidase. beta (Cerezyme)** Platelet Mab Fab (Reopro) **Annual sales over 500 million MANUFACTURER Janssen/J&J Janssen/J&J Genzyme/Sanofi Genzyme/Sanofi Lonza for Eli Lilly Bayer Merck-Serono Novartis Janssen/J&J Merck-Serono Genzyme/Sanofi Janssen/J&J Source: 11 th Annual Report and Survey on Biomanufacturing Capacity and Production, April 2014 Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August 2014, pg. 13. 17

Selected Continuous Bioprocessing Benefits Reduction in facility size, manufacturing footprint, etc. Significant costs savings, particularly investment in facilities Increases in flexibility No scale-up of bioprocesses Increased process robustness Less manual interactions Less bulk fluid input Less sensor insertions and other incursions into the process Increased automation PAT and upfront bioprocess design using QbD can be easier to implement Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August 2014, pg. 13. 18

Protein A Cell Wall Component of Several Strains of Staphylococcus aureus It is a Single Polypeptide Chain with Molecular Weight of 42,000 Daltons Specifically Binds to the Fc Region of Immunoglobulin Molecules Four High Affinity Binding Sites per Molecule 21

Industrialization of mab production technology. The bioprocessing industry at a crossroads. B. Kelley. mabs, (2009) Vol. 1, Issue 5, 443-452 26

Production capacity estimates for mammalian cell-derived mabs a Year CMO Product company Total Capacity at 2 g/l Capacity at 5 g/l 2007 500 kl 1,800 kl 2,300 kl 70 tons/yr 170 tons/yr 2010 700 kl 2,700 kl 3,400 kl 100 tons/yr 255 tons/yr 2013 1,000 kl 3,000 kl 4,000 kl 120 tons/yr 300 tons/yr a Capacity estimates from ref. 8. Industrialization of mab production technology. The bioprocessing industry at a crossroads. B. Kelley. mabs, (2009) Vol. 1, Issue 5, 443-452 27

Quality Control of MAbs The Challenges Large Complex Molecules Complex Manufacturing Systems Long Production Times Raw Material Concerns Stability of Raw Materials, Cell Lines, Intermediates, Drug Substance and Drug Product Changes and Comparability Immunogenicity Biosimilar MAbs 30

QC Testing of Monoclonal Antibody QC Release Products Cell Culture Potency Bioassay Antigen Binding Assay + ADCC if Mode of Action has Fc effector function implicated Characterization of Reference Material and Comparability 31

Cell Based ELISA 2 Ab CanAg + Tumor Cell Poly-lysine Alkaline phosphatase 2 Ab - goat anti-human IgG

Example: QC Certificate of Analysis for a Monoclonal Antibody Drug Substance Parameter Test Methodology Typical Specification Identity Peptide Map Consistent with Reference Material (retention time &% area comparison) Oligosaccharide Profile Consistent with Reference Material (X % G0, G1 etc.) Purity SDS-PAGE or CGE Consistent with Reference Material (Specify % distribution and ~ MWt.) Monomer Content by SEC IEF Residual Host Cell Proteins Ion Exchange Chromatography 95% IND; 98% BLA 1.5% Aggregate; 0.5% Fragment Consistent with Reference Material (Specify % distribution and ~ pi) 100 ppm (< 0.0001%)* (IND) Consistent with Reference Material (retention time &% area comparison)

Example: QC Certificate of Analysis for a Monoclonal Antibody Drug Substance Parameter Test Methodology Typical Specification Purity (cont.) Residual DNA 1 ppm *(IND) Potency Residual Protein A Other process related residuals Antigen Binding ELISA or SPR Cell Culture Bioassay 10 ppm* (IND) Depends on analyte measured 50-150% IND; 75-125% BLA 50-150% IND; 75-125% BLA Compared to Reference Material Safety Endotoxin <5Eu/mL FDA guidelines Bioburden Sterility NMT 1 CFU/mL Strength Protein Content by A280 As dictated by process Product Quality ph ± 0.1 ph unit Color and Appearance Excipients Describe solution properties Quantitative Determination

Types of Changes Cell Line / Culture Conditions Scale / Site Methods Purification Scheme Formulation, Storage, Dosage, Delivery Route and Systems Components / Vendors / Suppliers /CMO 35

Frequently Cited Concerns Animal Derived Components Aggregate Levels Immunogenicity Single Source Comparability Reprocessing and Retesting Validation Deficiencies Capacity 36

Antibody Fragmentation 37

Example Antibody Fragments Approved Products 1. LUCENTIS INN Name: Drug Substance: Manufacturer: Production Process: Manufacturing changes Implemented: Ranibizumab IgG1, anti-(hvegf) Fab fragment (humanmouse monoclonal rhu Fab V2 Ɣ1-chain), disulfide with human-mouse monoclonal rhu Fab V2 Κ-chain Genentech E-coli fermentation, homogenization, heat hold, dilution/ph adjustment (if necessary), centrifugation, filtration, 4 chromatography steps, centrifugation, UF/DF, dilution, buffer adjustment, formulation Cell line, cell culture conditions, fermentation process parameters, purification process, finished product formulation

Example Antibody Fragments Approved Products (cont.) 2. CIMZIA INN Name: Drug Substance: Manufacturer: Production Process: Manufacturing changes Implemented: Certolizumab pegol Recombinant, humanized, Fab' fragment with specificity for human TNFα conjugated to polyethylene glycol (PEG) via a maleimide group Drug substance is manufactured by Sandoz GmbH for UCB E-coli fermentation, primary isolation, purification, PEGylation Site, scale, manufacturing process changes (primary recovery, downstream processing) 39

Figure 1: Primary mechanism of action of antibody-drug conjugates: targeted delivery of a potent cytotoxic agent to cause cell death. BioPharm International, February 2013 40

Micro View of ADC 41

Why Make Antibody-Drug Conjugates (ADCs)? MAbs have exquisite selectivity and affinity for their target antigens They are not always effective as standalone therapies, but are excellent targeting modalities MAbs may thus serve as delivery vehicles to selectively deliver agents (toxins, radionuclides, imaging agents) to specific tissues Much current interest in delivering cytotoxics to cancer tissue via targeting tumor-selective antigens 43

Challenges in Development of Antibody Drug Conjugates Toxic molecule must stay attached to Mab while in circulatory system but be released after introduction into the cell. Conjugated Mab must retain the high affinity for the tumor cell antigen. The toxin must be released from the Mab, internalized, and then achieve a sufficient intracellular concentration to promote cell death. The potency of the released toxin must be high enough to kill tumor cells at low concentrations. 44

Challenges in Development of Antibody Drug Conjugates (cont.) Free toxin may be extremely dangerous due to high level of potency Difficult to work with in manufacturing and QC operations Special production facilities may be needed Design of linker, stability and conjugation scheme may be difficult to achieve. 45

The MAb - Considerations Can access fully human antibodies against desired target antigens with current technology (transgenic animals, phage display) May possess cytotoxicity alone (CDC, ADCC) Target antigens are tumor-selective, not tumor-specific! Selectivity, affinity 46

The Payload - Considerations Toxins utilized in current ADCs are highly cytotoxic 100x more potent than first generation ADCs Delivery must be well-controlled or there will be systemic toxicity importance of linker design Two general classes of toxin: Inhibitors of microtubule polymerization Molecules that damage DNA 47

FDA Approved ADC: TRASTUZUMAB + DM1 Kadcyla. http://www.rxlist.com/kadcyla-drug.htm 48

Recent Advances in the Biotechnology Industry Monoclonal Antibodies Fully humanized products Exploitation of antibody fragments with favorable characteristics Novel scaffolds introduced Glyco-engineering of antibodies and fusion proteins First antibody-drug conjugates approved 49

Acknowledgements Academic/Industry Colleagues: Dr. Marcia Federici Dr. Krish Venkat Former Graduate Student: Dr. Greg Hamilton Executive Administrative Assistant: Ms. Susan Mocko 50

Muito Obrigado! Perguntas? Antonio Moreira, Ph.D. Vice Provost for Academic Affairs University of Maryland Baltimore County 1000 Hilltop Circle Administration Building, Room 1001 Baltimore, MD 21250 Tel: (001) 410-455-6576 Fax: (001) 410-455-1107 Mobile: (001) 443-254-3696 Email: moreira@umbc.edu 51