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

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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

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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

S-S

S-SS-S

S-S

Carbohydrate

VH

VL

CL

CH1

CH2

CH3

Intrachain disulfide bonds

Hinge region

L Chain hypervariable regions

H Chain hypervariable regions

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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.

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Complex Carbohydrate in Antibody Structure

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BioPharm International, February 2013

Figure 2: List of FDA-approved antibody therapeutics.

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Thayer, C&EN Houston. cen.acs.org. October 7, 2013.

KEY TARGETS Developers are trying to create functional replicas of leading biologic drugs.SOURCES: Company data, Biotechnology Information Institute

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Herceptin Binding to HER2 Receptor

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

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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

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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.

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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.

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Bioreactor Operating Modes

• Batch • Fed-Batch • Continuous • Perfusion

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Table 1: Selected Perfusion Operations Issues: PRODUCT MANUFACTURER

IL-12/23 Mab (Stelara)** Janssen/J&J

TNF Mab (Simponi) Janssen/J&J

Glucosidase alfa (Myozyme)** Genzyme/Sanofi

Galactosidase alfa (Fabrazyme)** Genzyme/Sanofi

Protein C (Xigris) Lonza for Eli Lilly

Factor VIII (Kogenate-FS)** Bayer

Interferon beta (Rebif)** Merck-Serono

IL-2 receptor Mab (Simulect) Novartis

TNF mAb (Remicade)** Janssen/J&J

FSH (Gonal-F) Merck-Serono

Galactosidase. beta (Cerezyme)** Genzyme/Sanofi

Platelet Mab Fab (Reopro) Janssen/J&J **Annual sales over 500 million

Source: 11th Annual Report and Survey on Biomanufacturing Capacity and Production, April 2014

Continuous Bioprocessing and Perfusion. E. Langer. Pharmaceutical Processing, July/August 2014, pg. 13.

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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.

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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.

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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.

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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

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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.

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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.

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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.

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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.

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

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Production capacity estimates for mammalian cell-derived mAbsa

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

aCapacity estimates from ref. 8.

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

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

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

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

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QC Testing of Monoclonal Antibody Products

• QC Release – Cell Culture Potency Bioassay – Antigen Binding Assay – + ADCC if Mode of Action has Fc effector

function implicated • Characterization of Reference Material and

Comparability

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CanAg + Tumor Cell

Poly-lysine Alkaline phosphatase

Cell Based ELISA

2° Ab

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 ≥ 95% IND; ≥98% BLA ≤ 1.5% Aggregate; ≤ 0.5% Fragment

IEF Consistent with Reference Material (Specify % distribution and ~ pI)

Residual Host Cell Proteins ≤ 100 ppm (< 0.0001%)* (IND) Ion Exchange Chromatography 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) Residual Protein A ≤ 10 ppm* (IND) Other process related residuals

Depends on analyte measured

Potency Antigen Binding ELISA or SPR

50-150% IND; 75-125% BLA

Cell Culture Bioassay 50-150% IND; 75-125% BLA Compared to Reference Material

Safety Endotoxin <5Eu/mL FDA guidelines Bioburden NMT 1 CFU/mL Sterility

Strength Protein Content by A280 As dictated by process Product Quality pH ± 0.1 pH unit

Color and Appearance Describe solution properties Excipients 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

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Frequently Cited Concerns

• Animal Derived Components • Aggregate Levels • Immunogenicity • Single Source • Comparability • Reprocessing and Retesting • Validation Deficiencies • Capacity

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Antibody Fragmentation

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Example Antibody Fragments Approved Products

1. LUCENTIS® INN Name: Ranibizumab Drug Substance: IgG1, anti-(hVEGF) Fab fragment (human-

mouse monoclonal rhu Fab V2 Ɣ1-chain), disulfide with human-mouse monoclonal rhu Fab V2 Κ-chain

Manufacturer: Genentech Production Process: E-coli fermentation, homogenization, heat

hold, dilution/pH adjustment (if necessary), centrifugation, filtration, 4 chromatography steps, centrifugation, UF/DF, dilution, buffer adjustment, formulation

Manufacturing changes Cell line, cell culture conditions, Implemented: fermentation process parameters,

purification process, finished product formulation

Example Antibody Fragments Approved Products (cont.)

2. CIMZIA® INN Name: Certolizumab pegol Drug Substance: Recombinant, humanized, Fab' fragment with

specificity for human TNFα conjugated to polyethylene glycol (PEG) via a maleimide group

Manufacturer: Drug substance is manufactured by Sandoz GmbH for UCB

Production Process: E-coli fermentation, primary isolation, purification, PEGylation

Manufacturing changes Site, scale, manufacturing process changes Implemented: (primary recovery, downstream processing)

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BioPharm International, February 2013

Figure 1: Primary mechanism of action of antibody-drug conjugates: targeted delivery of a potent cytotoxic agent to cause cell death.

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Micro View of ADC

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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

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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.

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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.

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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

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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

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Kadcyla. http://www.rxlist.com/kadcyla-drug.htm

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FDA Approved ADC: TRASTUZUMAB + DM1

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

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Acknowledgements

Academic/Industry Colleagues: Dr. Marcia Federici Dr. Krish Venkat

Former Graduate Student: Dr. Greg Hamilton

Executive Administrative Assistant: Ms. Susan Mocko

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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

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