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
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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
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
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
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
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