MONOCLONAL ANTIBODY DEVELOPMENT AND...

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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Apresentador

Notas de apresentação

Batch; Simplest of the three, Low cost, Low yields, Quick turnaround, Designed to be quick and efficient Choose media, sterilize, inoculate and harvest Fed-batch; More difficult to perform, Higher yields, Requires more supervision, Moderate time to perform Designed to increase production and yields Choose media and nutritional additions, inoculate, monitor substrate level, add nutrients, harvest Continuous; Most difficult to perform; Highest yields; Constant supervision; Large time investment Designed to run for long lengths of time with high yields, with one inoculation Choose media, inoculate, feed new media while removing older media and harvesting

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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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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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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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759494/#R8

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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: [email protected]

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