GENETICALLY MODIFIED ORGANISMS FOR MEDICINES PRODUCTION

Antonio Moreira University of Maryland Baltimore County III SYMPOSIUM SINDUSFARMA-IPS/FIP-ANVISA New frontiers in manufacturing technology, regulatory sciences and pharmaceutical quality system Brasilia August 5, 2014

Presentation Outline

• Primer on genetic engineering and biotherapeutics • Main genetically modified organisms currently used in biomanufacturing • Biomanufacturing processes • Examples of bioprocess development studies • Status of the biotech industry for therapeutic products

How Do We Deliver & Maintain Our Protein Coding Sequences?

Plasmids: Autonomously replicating, circular DNA molecules Circular plasmids can be transfected into cells directly or plasmids can be linearized.

Selections used include antibiotics, puromycin, hygromycin, neomycin, presence of DHFR gene (MTX r ) Drug level can be used to select for increased numbers, or copies of gene. 4

Clark & Pazdernik. Biotechnology: Applying the Genetic Revolution. 2009.

Biologics As Therapeutics

• Natural Products • Blood Products - Transfusions • Vaccines • Purified Proteins from natural sources / tissues • Recombinant Proteins – Microbial – Cell Culture • Monoclonal Antibodies • Combination Products • Cell Therapy – Bone Marrow Replacement / Reconstruction • Stem Cell Therapy 6

Figure 1. Total sales in the US and Europe of traditional pharmaceuticals
(blue) and biopharmaceuticals (green) are shown by year for the past decade.
Sales information was obtained from company annual reports and other publically available sources.

Jones & Ecker. Pharmaceutical Processing, October 2013.

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Figure 2: List of FDA-approved antibody therapeutics.

BioPharm International, February 2013 8

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 9

Bioprocesses Mirror the Complexity of Biological Products

• Classical Pharmaceutical Drugs – Defined Structure and Characteristics • Basics of biopharmaceuticals – Complex chemistry and structures – Proteins, nucleic acids, lipids, polysaccharides – Cells • Inherently unstable (until purified or formulated) – Degradative enzymes co-produced – Temperature, pH, concentration • Bio-safety constraints - complexity and purity • Process design - control for productivity • Process design for GMP vs. flexibility 10

NCE Versus Biopharm Comparison

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Figure 1: Global biosimilar approvals, 2006-2012 (in Europe, unless otherwise indicated.

Emerton, Duncan. Supplement to BioProcess International. June 2013.

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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.

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Biomanufacturing • Biomanufacturing involves three key processes:

– Controlled growth of microorganisms, cells, tissues or organisms – Conversion of simple raw materials or complex molecules to desired product – Isolation & purification of the product from complex mixtures 14

Major Production Systems • Bacterial Cells

– E. coli

• Yeast Cells

– – Saccharomyces cerevisiae Pichia pastoris

• Mammalian Cells

– Chinese Hamster Ovary – CHO – Baby Hamster Kidney –BHK – NS0 or Sp2/0 (Mouse Myeloma) 15

Subcellular Structure of bacteria are approximately 0.6 microns by 1-2 microns. Courtesy of Rocky Mountain Laboratories, NIAID, NIH.

Escherichia coli : Scanning electron micrograph of E. coli . The rod-shaped Clark, D. & Pazdernik, N.

Biotechnology; Applying the Genetic Revolution

. 2009 16

Major Protein Expression Platforms

• Yeast Cells/Fungi Pichia, Saccharomyces, Kluyveromyces, Aspergillus

Pichia spp.

Saccharomyces spp.

Aspergilus spp.

Clark, D. & Pazdernik, N.

Biotechnology; Applying the Genetic Revolution

. 2009 18

Clark & Pazdernik. Biotechnology: Applying the Genetic Revolution. 2009.

Figure 1: Industrial cell-free biology.

Swartz, AIChE Journal, January 2012. Vol 58, No. 1.

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Fig. 2. Cartoon comparison of in vivo recombinant DNA protein expression with cell-free protein synthesis (CFPS). CFPS systems provide a more rapid process/product development timeline. Example proteins shown include a virus-like particle (VLP), single-chain antibody variable fragment (scFv), and a membrane bound protein (MBP).

Carlson, ED, et al, Cell-Free Protein Synthesis: Applications Come of Age. Biotechnol Adv. (2011).

Swartz, AIChE Journal, January 2012. Vol 58, No. 1.

Mammalian Antibody Production – Cell Culture

Media Prep

HEAT COOL

Media Pasteurizer Vial Thaw / Inoculum Expansion 50-Liter 500-Liter

Upstream Processes

5000-Liter 20,000-Liter

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Mammalian Antibody Production – Harvest

Harvest from Production Bioreactor

Transfer to Purification Suite Virus Inactivation

rProtein A affinity chromatography

Depth Filter Disc-Stack Centrifuge

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Mammalian Antibody Production – Downstream Processing

Ion (Cation) Exchange Chromatography Intermediate Storage Ion ( Anion) Exchange Chromatography Intermediate Storage Viral Filtration

20m

2 API I.B.I

CryoPreservation System Bulk Filtration (BDS) UF/DF Step

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Intermediate Storage Hydrophobic Interaction Chromatography

Stirred-tank Reactor

• Most common type • Cylindrical tank • Agitator motor, shaft & impellors • Air/gas inlet & exhaust • Sampling & harvest ports

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Example of a Commercial Bioreactor

• Sandoz cell culture manufacturing facility • Located in Schaftenau, Austria • Facility contains two 13,000 L bioreactors 27

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Supplement to Pharmaceutical Engineering, 2013, p.10-16 29

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)** TNF Mab (Simponi) Janssen/J&J Janssen/J&J Glucosidase alfa (Myozyme)** Galactosidase alfa (Fabrazyme)** Protein C (Xigris) Factor VIII (Kogenate-FS)** Interferon beta (Rebif)** IL-2 receptor Mab (Simulect) TNF mAb (Remicade)** Genzyme/Sanofi Genzyme/Sanofi Lonza for Eli Lilly Bayer Merck-Serono Novartis Janssen/J&J FSH (Gonal-F) Galactosidase. beta (Cerezyme)** Platelet Mab Fab (Reopro) **Annual sales over 500 million 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.

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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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Typical Downstream Processes

• Cell Lysis/Disruption (if needed) • Chromatographic purification • Product Concentration • Sterile Filtration • Formulation • Fill & Finish 33

Purification

• Sequence of steps, generally 3 to 4 • Selective removal of contaminants • Isolates molecules by physical or chemical characteristics • Volume reduced at each step, ideally • Initial coarse cuts • Polishing steps 34

Production Scale Units

Purity Goals

• Residual host & contaminant proteins – ppm level • Nucleic acids – 100 pg/dose • Viruses – Below detection limits • Endotoxins – < 5EU/kg/hr for parenteral use; <0.25 EU/kg/hr for intrathecal use for drug products w/o a compendial limit • Microorganisms – None 36

Regulation of New Technology Protein Production Issues

• Expression Technology – Cell line selection and optimization – New production systems to increase yield • Bacteria – newer strains 10 g/L • Yeast – over 5 g/L • Mammalian Cells – achieving 2-5 g/L • Recovery & purification – Product aggregation – Solubility – Stability • Characterization of complex structures – Glycosylation – Pegylation • Process and Facility design – Automation – Cleaning 37

• Past Experience • Preclinical Studies • Clinical Studies

Product Realization

Design Space

Determining What and How the Process Affects Product Characteristics • Process Control Strategy • Testing • Quality Systems Identification of Key Product Characteristics

Target Product Profile

• Past Experience • Process Development • Product Characterization Ensuring Patient Receives the Expected Product

Quality Assurance

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Discovery to Therapeutic Delivery Pathway and the Process Information Gap

Phase

Discovery

Issues

Product Characterization

Non-Instrumented Fully Instrumented

Process optimization Define Design Space Production Scale Quality Control Therapeutic delivery Fundamental question: Can we predict manufacturing behavior of cell lines as early as in non-instrumented devices? Discovery phase is not integrated with following phases. It lacks of the “know why” process perspective.

Based on Kirouac and Zandstra 2008, Cell Stem Cell 3:369-381

PSDs Applications in Upstream Bioprocess Development and the Process Information Gap

STRAIN/CLONE SCREENING SCALE-UP EARLY R&D STUDIES PROCESS OPTIMIZATION PRODUCTION

Fully instrumented Non-instrumented

Static + Stir

MCB vial

Stir Shake Roller Wide variety of PSDs.

SCALE-DOWN

Application of Process Analytical Technology for Extended Cell Passaging: A Proof-of-Concept Revealing Study MCB vial Expression system: Non-adherent SP2/0-based myeloma/ mouse (2055.5) Protein: IgG3 antibody specific for the Nisseria meningitides capsular-polysaccharide (MCPS).

Media: CD Hybridoma GT TM 20mL CO 2 Incubator 5% CO 2 37 O C 3-Day passage scheme P P P i+1 1,1 4,2 , j , P , P 2,1 5,2 , P , P 3,1 6,2 P i+3 , j+1 P i+n,j+n’ i: Number of passages j: Number of T-flasks i+n: Total number of passages j+n’: Total number of T-flasks Vallejos et al. (2010) “Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask” Biotechnol & Bioeng. 105(6):1040-1047

Application of Process Analytical Technology for Extended Cell Passaging: A Proof-of-Concept Revealing Study  Cells are exposed periodically to sub-optimal DO levels (0%).

 What happened at passages 18-20 and passages 27-29?

Vallejos et al. (2010) “Dissolved oxygen and pH profile evolution after cryovial thaw and repeated cell passaging in a T-75 flask” Biotechnol & Bioeng. 105(6):1040-1047

Application of Process Analytical Technology for Extended Cell Passaging: A Proof-of-Concept Revealing Study Improving cell passaging techniques in T-flask at low cost!

100 80 60 40 20 8.5

0 8.0

7.5

T75-Static T75-Rocking 7.0

Vallejos et al. (2012) Biotechnol & Bioeng. 109 (9):2295-2305

6.5

 Sub-optimal DO and pH levels are avoided in rocking T-flasks.

6.0

0 20 40 60 80 Cell Culture time (h)

Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305

100 120

A Novel Scale-Down Paradigm for the Wave Bioreactor

Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305 44

A Novel Scale-Down Paradigm for the Wave Bioreactor

 At matched k L a both systems (rocking T-flasks and wave bioreactor) perform similar except for specific productivity Vallejos et al. (2012) “Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools” Biotechnol & Bioeng. 109(9):2295-2305 45

Comparability Study 5L Vs. MB

     Same seed in both systems; Passage 6-10 DO 30% pH 7.2, Control w/o base addition Matched Kla 3 replicates 46 H/D Impeller type Number of impellers Overlay rate Kla hour -1 DO set point pH set point Temp.

Rpm

Base line comparability study 5L MB

1.5

2.2

pitched blade pitched blade 2 0.03vvm

3 0.03vvm

2 30% 7.2

2 30% 7.2

37 0 C 220 37 0 C 220

Difference in the Antibody Product Titer

The antibody titer in 5L was about 50% higher than that in the minibioreactor 47

Glutamine Profile in 5L Vs. Minibioreactor

  Glutamine degradation was not significant L- Glutamine levels reach zero when minibioreactors reach their stationary /peak cell density phase, while the 5L has sufficient amount of glutamine 48

Glutamine Supplement Experiment

Glut MB control Glut MB Supplemented Titer MB Control Titer MB Supplemented Titer 5L   Protein titers were similar to 5L when glutamine was supplemented Identical amount of starting glutamine concentration Difference in the glutamine consumption 49

Comparability Study With CO

2

Monitoring

( Source: Ge et al., 2005)

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Comparability at Similar CO

2

Rates Stripping

The CO 2 profiles of minibioreactor (MB) and bench scale bioreactors (5L) were found to be similar 51

Result of CO

2

Profile Matching

H/D Impeller type Mixing time overlay rate mixing time Kla per hour CO 2 Stripping rate ppm/min Peak VCD (X10^6cells/ml) Peak titers (mg/L) G0F G1F G2F

5L

1.5

Base line study

MB

2.2

pitched blade 4S 0.03vvm

4 S 2 pitched blade 4S 0.03vvm

4 S 2 298 ± 4 145 ± 4 4 ± 0.8

178 ± 12 42.5

± 5.5

48.3

± 2.4

7.4

± 2.5

4.5

± 0.8

122 ± 8 48.3

± 0.9

44.3

± 0.8

7.4

± 0.2

After providing similar CO 2 stripping rates

5L

1.5

MB

2.2

pitched blade 4S 0.03vvm

4 S 2 pitched blade 4S 1.1 vvm 4 S 2 298 ± 4 223 ± 4 4.4

163 ± 12 45.2

± 1.4

45 ± 4.2

9.9

± 2.9

5.1

± 0.8

164 ± 8 46.5

± 0.9

42.1 ± 1.6

11.2

± 0.8

  Protein titers in MB with similar CO 2 found to be similar to that in 5L stripping rate were A difference in the CO 2 difference in the titers profile could be a reason for a 52

Productivity of IgG3 at varying %DO. Concentration of IgG3 samples was measured by A280nm.

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Comparison of averaged relative protein carbonyl concentration measured by ELISA in HTBRs at various %DO (10%, 40%, 60% and 80%). Error bars represent standard deviation of protein carbonyl content.

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Current and Novel Inputs to Bioprocess Scale-down and Evaluation

New Approach Sentinel genes showing cell physiological response Pre-culture

tunable bioprocess parameters P/V, k L a, t mix

Culture growth

parameters - X V , µ, q oxygen , q Ammonia p, Glu, Gln, Lac,

Real-time

process set points for %DO, pH, T, pCO 2

Scale-up/ Scale down

5L bench-scale to Mini bioreactors  New approach to bioreactor Scale-change  Lack of measurable attributes at cellular level 55

Post-culture

product quality Titer, Purity, Glycosylation

Recent Advances in the Biotechnology Industry

• • • Mammalian Cells • Significant increase in manufacturing scales • • Major product titer improvement (up to 8-10 g/l) Large gains in recovery yields • • API batch sizes of up to 50 to 100 KG Perfusion processes up to 1000L scale with high perfusion rates and lasting up to 200 days 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 Innovation in Process and Product Development • Maturation of technologies and processes • • Higher degree of automation Process robustness driven by science and process management 56

Recent Advances in the Biotechnology Industry (Cont.)

• • • • Process Development • Improved cell lines • Enhanced media compositions and media optimization • Improved chromatographic media • • Improved expression vectors Use of “omics” approaches Implementation of Disposable Technologies • Many components available • Smart integration of mature technologies • Fully disposable facilities Increased Efficiencies and Risk Mitigation • Elimination of animal-derived raw materials • • • Implementation of virus clearance Implementation of platform bioprocess technologies Pursuit of PAT and QbD initiatives Pharmaceutical R&D • Formulation, manufacture and delivery of biological drug products • • • • Development of stable high concentration formulations Use of pre-filled syringes Development of auto-injectors Development of combination products • Aseptic processing technologies 57

Expectations for the Next Decade

• • • • • • • • • • • Development of combination products Increases in automation and paperless manufacturing Biopharmaceutical manufacturing facilities • Platform based large scale for large market regions • Disposable plants in emerging markets Focused attention on QbD and PAT objectives Increased downstream process efficiency Enhanced physical characterization of bioreactors • • Mass transfer (especially CO 2 ) Mixing • Shear forces Renewed focus on traditional engineering principles • • • Technology transfer Process scale-up Modeling unit operations (i.e., CFD) including Artificial Neural Networks and Statistical Online Control Continuous processing New molecular formats Evolving regulatory requirements Global access to high quality biopharmaceuticals 58

Acknowledgements

Academic/Industry Colleagues: Dr. Marcia Federici Dr. Debra Barngrover Dr. Govind Rao Former Graduate Students: Dr. Bhargavi Kondragunta Dr. Jose Vallejos Dr. Shaunak Uplekar Ms. Nacole Lee Executive Administrative Assistant: Ms. Susan Mocko

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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