Biologic Usa 10 2004

Plant-Based Manufacturing of Biosimilar and Next-Generation Biologics

Daniel Tusé, Ph.D. Vice President, Business Development Large Scale Biology Corporation 19 October 2004 3333 Vaca Valley Parkway, Vacaville, CA 95688, USA. Tel. 707 446 5501. Fax. 707 446 3917. Web. www.lsbc.com

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Large Scale Biology Corporation ƒ Presentation outline - Technology/platform background - Applications - Case study: Development of a follow-on product FTO CMC/product quality - Lessons learned: Regulatory experience

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Large Scale Biology Corporation ƒ Product-focused biopharmaceutical company ƒ Strengths: Development Î Manufacturing ƒ Areas: Therapeutic proteins and vaccines

Vacaville, California Headquarters; Product Development

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

Owensboro, Kentucky Process Development; cGMP Biomanufacturing

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Large Scale Biology Corporation (LSBC) ƒ GENEWARE® plant viral gene expression & biomanufacturing platform ƒ Development of proprietary and partner-specified molecules - Cancer and antiviral vaccines - Enzymes for replacement therapy - Animal health vaccines & therapeutics - Next-generation biologics (improved Type I IFN variants) ƒ Biomanufacturing of follow-on molecules - r-Aprotinin - r-Interferon alpha 2a and 2b © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Industry Preferences – Biomanufacturing of FOBs ƒ Adopt the innovator’s manufacturing method + Process is familiar to regulatory agencies + Should be able to supply to meet market demand + Product should reproduce innovator’s specifications + Impurity profile should be similar to innovator’s – API/impurity profiles may not be the same as innovator’s – FTO: No compositional patent coverage does not mean no process patent coverage – FTO in new markets possible; may not access prime markets © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Potentially Blocking US Process Patents for r-INF-alpha US Patent No.

Assignee

Comments

1 2 3 4 5 6 7

Genentech Genentech Genentech Toray PBL Biomed. Genentech Genentech

Microb. production of rhIFN Microb. production of rhIFN Microb./animal IFNs IFN stabilization/E. coli Fusion prots./rhIFNs Porcine & bovine IFNs DNA & vectors for IFNs of non-human origin/E. coli Vector & process for expr. IFNs in E. coli Yeast vectors for rIFNs rIFN made in E. coli; purific. by affinity chromatography

8

6,610,830 6,482,613 6,432,677 6,391,296 6,225,455 5,831,023 5,827,694 5,710,027

9 5,646,037 10 5,196,323

Boehringer Ingelheim Ciba-Geigy Boehringer Ingelheim

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

Expiration 2020 2019 2019 2019 2018 2015 2015 2015 2014 2010

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Potentially Blocking US Process Patents for rh-G-CSF US Patent No.

Assignee

Comments

1

6,521,449

GSF

Microb. production of rh-G-CSF

2019

2

5,599,449

Amgen

Microb. production of rh-G-CSF

2014

3

5,874,075

Amgen

Stabilization/E. coli rhG-CSF

2016

4

5,840,543

ICI

Microb. production of rhG-CSF

2015

5

5,472,857

Amgen

Microb. produced rcG-CSF

2012

6

5,284,456

Amgen

Pulmonary delivery of E. coli-made rh-G-CSF

2011

7

4,810,643

Amgen

Pluripotent G-CSF made in E. coli or in a eukaryotic cell

2006

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

Expiration

LSBC PROPRIETARY – 41006 - 7

GENEWARE® Technology Platform Gene Expression Using RNA Viral Vectors Advantages from what they are… as well as what they aren’t … -

Tobacco Mosaic Virus (TMV) and related viruses Members of the Alphavirus superfamily RNA (+) stranded genome (“messenger RNA”) Extranuclear replication and protein expression Encode only 3 to 4 native proteins No cell membrane, cell wall, cytoplasm, organelles, etc. Propagate in non-transgenic, non-food/feed hosts Safe (environmental containment, human exposure)

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GENEWARE® Tobacco Mosaic Virus (TMV) Soluble (Free) Protein Viral Vector Cap structure

Replicase / transcriptase genes

RNA single plus (+) strand viral genome

Product gene

Movement protein gene

Coat protein gene

In plant hosts, the movement protein translocates virions systemically to ensure high yield

>2,100 coat proteins per virion stabilize the RNA genome

Product of interest is produced through subgenomic promoter and is produced as soluble protein, which accumulates within plant cells, or is secreted into the apoplast (interstitial fluid), for flexibility in extraction and purification Schematic showing part of the Tobacco Mosaic Virus (TMV) rod-shaped virion.

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

Wide variety of soluble (free) protein products produced with high efficiency LSBC PROPRIETARY – 41006 - 9

Virus-Like Particle (VLP) TMV Coat Protein Fusion Viral Vector Cap structure

Movement protein gene

Replicase / transcriptase genes

RNA single plus (+) strand viral genome

In plant hosts, the movement protein translocates virions systemically to ensure high yield

N-terminal fusion Loop region fusion C-terminal fusion

Peptides, fused at the gene level to the coat protein of TMV, are arrayed for display on the viral surface. Over 2,100 peptides can be displayed on the surface of each virion, forming hybrid TMV-peptide Virus-Like Particles (VLP) which can be efficiently manufactured and purified.

Coat protein gene fused to peptide at one of three locations

RNA helix

loop

N terminus

C terminus

Schematic showing part of the Tobacco Mosaic Virus (TMV) rod-shaped virion.

Schematic showing two adjacent coat proteins and N-, Loop- and C-terminal fusion options for displaying peptides on the TMV virion surface.

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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LSBC’s Expression Vectors Cap structure

Movement protein

Replicase

Product (eg. GFP) Coat

Normal Light

2 dpi

4 dpi

6 dpi

Product

(eg. GFP) UV Light © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Plant Cell Structure

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Targeting of Polypeptide to Intracellular Compartments

Non-Specific and Specific Accumulation of Protein in Selected Plant Cell Compartments Enables Rational Product Extraction and Purification © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Targeting of Polypeptides To The Apoplast Downstream Processing Optimization Plants secrete only ~1% of their total cellular protein into the interstitial space between cells

Apoplast targeting enables product concentration in a fluid devoid of most host proteins

Apoplast (interstitial space) © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

Recovery of Protein by Vacuum Infiltration Very little carryover of host proteins into the API stream Very simple purification Dramatic reduction in COGS Proteins so recovered - Are correctly folded - Meet purity specs - Have entered the clinic LSBC PROPRIETARY – 41006 - 14

Features of Viral Expression ƒ Speed

Production cycle is 2-3 weeks duration (indoors) - No plant breeding required

ƒ Copy Number Tens of thousands of copies of transcript per cell - Chloroplast transformation: 10,000 cpy/cell - Nuclear transformation: 1-2 cpy/cell ƒ Purification

Product can accumulate in plant cell compartments, or on the vector itself - More flexible extraction/purification options

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Features of Viral Expression (continued) ƒ Containment

TMV viral vectors are not transmissible by - Pollen - Seed - Insect/arthropod vectors Viral vectors are not persistent in soil Host plants used are Nicotiana species - Not food or feed crops - Environmental, food/feed contamination are not issues

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Features of Viral Expression (continued) ƒ Safety

No animal-sourced raw materials, components or additives used in the manufacture of APIs (shared with other PMP systems) APIs manufactured via viral vector technology are free of potentially problematic plant- and vectorderived contaminants

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Indoor Manufacturing: Greenhouse Complex

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Field-Scale Protein Manufacturing

Outdoor cultivation and viral inoculation under USDA permits Policy clearances to 1,000 acres © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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LSBC’s Commercial-Scale Facility for cGMP Biopharmaceutical Manufacturing

LSBC Biomanufacturing Campus, Owensboro, Kentucky, USA Facility: 30,000 sq ft. Throughput: 3 metric tons of plant biomass/hr © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Biomass Homogenization

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Extract Clarification

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Clarification

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Chromatography/Ultrafiltration

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Finish and Fill

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LSBC’s Product Development and Manufacturing All Manufacturing Process Modules and Components in Place Today Quality and Regulatory Quality Control Quality Assurance Quality Systems FDA Submissions Fill & Finish Class 100 Facility Sterile Vial Fill Product Purification Clarification Filtration Chromatography

Protein Expression Technologies

LSBC Biomanufacturing

Product Extraction Homogenization Interstitial Fluid

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

Analytical Development Activity Assays Product Identity Product Purity Process Development Bench-scale Pilot-scale Manufacturing-scale

Agronomics Field Crop Production Greenhouse Production Agriculture USDA Regulatory LSBC PROPRIETARY – 41006 - 24

US Regulatory Agency Oversight

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Summary: GENEWARE Environmental Releases ƒ First Recombinant Virus Release in 1991 ƒ Over 15 Releases, 3 States, 198 Acres ƒ Released Tested Over 17 Unique Products ƒ First Field Release of Genomic Libraries ƒ 2004: Of 44 Acres Approved Nationwide, LSBC had 35 (80% ) ƒ 2004: Used as Training Site for USDA Inspectors ƒ No Adverse Public Reaction

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Viral Expression of r-Aprotinin in Plant Hosts Protease inhibitor 6511 Da protein 58 amino acids 3 disulfide bonds Well characterized Activity from molecular properties alone ƒ Market expansion projected from existing and new indications, from current $180 M/yr (ann.) ƒ ƒ ƒ ƒ ƒ ƒ

ƒ Marketed product Bayer’s Trasylol® ƒ Bovine sourced

© 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

Phe

Tyr

Tyr

Asn

Arg

Ala

Ile Ile

Lys

Arg Val

Tyr

Ala

Gly

Lys

Phe

Gly

Cys

Asn

Cys

Pro

Gly

Thr Gln

Lys

Leu Cys

Ala

Arg

Ala

Gly

Phe

Ser

Asn

Arg

Ala

Asp

Glu

Cys Met

Lys

Arg

Thr Tyr Pro

Pro

Glu

Leu Phe

Asp Arg

Thr Cys Cys Gly Gly

Pro Ala

LSBC PROPRIETARY – 41006 - 27

r-Aprotinin Extraction and Purification Master Plasmid Bank

Plant Propagation

Chromatography Separations

Inoculation Working Plasmid Bank

Ultrafiltration and Diafiltration Plant Harvest and Extraction

Transcript and Virion Stocks

0.2 µM Filtration Clarification

Host Seed

Bulk Fill Ultrafiltration and Diafiltration

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Test Methods and Results of Aprotinin Comparisons Assay

Comparative Attribute

Identity by Tryptic Digest MALDI-TOF mass mapping

Conforms with bovine lung aprotinin predicted tryptic fragments and fragment derivatives (84% amino acid coverage)

Identity by MALDI-TOF MS

r-Aprotinin

Trasylol

Conforms

Conforms

6,512 Da ± 0.05%

6,512 Da

6,512 Da

Identity by Amino Acid Analysis

Conforms with bovine lung aprotinin amino acid composition

Conforms

Conforms

Purity by SDS-PAGE

Purity

>99%

>99%

Circular Dichroism

Identity/Structure

Comparable

Comparable

Purity by GC/MS Small molecular weight host toxicants

Purity

Comparable levels of target compounds

Comparable levels of target compounds

Purity by Appearance

Clear, colorless, free of visible particles

Clear, colorless, particle free

Clear, colorless, particle free

Potency by Specific Activity

>6,500 KIU/mg protein

7,145 KIU

7,143 KIU

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r-Aprotinin Biomanufacturing Summary Things you can control well … ƒ r-Aprotinin can be manufactured at scale (100s of Kg API per year) using transient plant viral expression ƒ The API conforms to the innovator product ƒ The impurity profile of r-Aprotinin is “the same but better” than the innovator product’s ƒ LSBC’s “Animal-Free” r-Aprotinin is currently sold for nonpharma applications (R&D, cell culture) through Sigma-Aldrich

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Pre-IND Meeting with FDA Things you can control less well … ƒ LSBC requested a pre-IND meeting with FDA to discuss the regulatory and clinical development path for a recombinant aprotinin product for CABG patients - Meeting was conducted in Q2, 2004 - Office and Division Director were in attendance - Meeting was attended by all review disciplines

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Purpose of the Pre-IND Meeting ƒ The primary purpose of the meeting was to discuss an abbreviated clinical program with the Agency - A well-developed clinical plan was presented - The clinical plan represented an “equivalence” approach (not a full efficacy program) ƒ The secondary purpose was to discuss an “abbreviated” regulatory approval route such as 505(b)(2) or 505(j)

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Purpose of the pre-IND Meeting ƒ From a product perspective, the primary purpose of the meeting was to determine if a well-characterized, recombinant aprotinin would be viewed by the Agency as a “follow-on” product to the bovine-sourced approved product Interestingly, the Agency was not focused on the difference in the source of aprotinin, rather the focus was on the ability to characterize the molecule

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r-Aprotinin Development Strategy ƒ LSBC proposed a small pilot clinical study, followed by a single Phase II trial to demonstrate PK, and a minimal Phase III ® program to demonstrate non-inferiority to Trasylol ƒ The Agency provided regulatory and clinical guidance, including its opinion that: - The API manufacturing process is defined and reproducible - The methods used to characterize the API are adequate - The proposed clinical program is appropriate in size, scope ® and endpoints to confirm equivalence to Trasylol

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Regulatory Issues and Guidance ƒ With regards to the 505(b)(2) approval pathway for recombinant aprotinin… The Agency recommended a 505(b)(1) route since there is currently no clear regulatory path for a “follow-on” therapeutic protein. HOWEVER,

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Regulatory Issues and Guidance [505(b)(2) continued] The Agency commented that a 505(b)(2) route relying on literature and the additional clinical studies proposed would be another option NOTE: The primary obstacle to 505(b)(2) is the inability to access innovator data

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Immunogenicity Issues and Guidance ƒ With regards to assessment of seroconversion and hypersensitivity rates in CABG patients, where larger number of patients would be appropriate, the Agency concurred with a post-approval assessment plan and patient registry

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Indication and Label Issues ƒ If a pivotal trial demonstrates non-inferiority to Trasylol®, and the safety is similar, the Agency commented that the same label indication would be applicable

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Comments Regarding “Follow-On Biologics” ƒ There is currently no regulatory path to accommodate “biogenerics” ƒ The Agency has begun developing guidance and policy for “follow-on biologics” and will have many open forums. This is in part due to pressure from Congress regarding the shortage and cost of new products (See Innovation and Stagnation Report)

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Comments Regarding “Follow-On Biologics” ƒ The Agency is being encouraged to find “examples” of products that may serve as models for “follow-on” development pathway ƒ The Agency has openly commented regarding immunogenicity and comparability issues around “follow-on biologics” development ƒ Aprotinin is a small protein of relatively low complexity ƒ The FDA concurred with an abbreviated pathway partly based on ability to manufacture and characterize this small protein ƒ There are numerous advantages and reasons for moving away from the bovine-sourced aprotinin

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Molecular Complexity and Regulatory Complexity

Simpler, More Easily Characterizable Molecules Will Likely Move Faster Through the Regulatory Process GM-CSF 127 / ~17,000 Da Molecule / No. aa / MW in Daltons Manufactured by LSBC Bivalirudin 20 / 2,180 Da Glucagon 29 / 3,483 Da Nesiritide 32 / 3,464 Da

Octreotide 8 / 1,019 Da Eptifibatide 8 / 832 Da Leuprolide 9 / 1,269 Da Desmopressin 9 / 1,183 Da

Enoxaparin ps / ~4,500 Da Insulins 51-54 / 5,800 – 6,100 Da Aprotinin 58 / 6,512 Da Lepirudin 65 / 6,979 Da

EPO 165 / 30,400 Da Interferon 166 / 22,500 Da G-CSF 175 / 18,800 Da scFv 240 / 27,000 Da Fab 450 / 52,000 Da hGH 191 / 22,125 Da Reteplase 355 / 39,571 Da Imiglucerase 497 / 60,430 Da tPA 527 / 58,000 Da Lysosomal Acid Lipase 378 / 50 kDa α-Galactosidase 400 / 101 kDa MAbs >1,000 / 145-155 kDa

Increasing molecular complexity: # of amino acids, MW, folding, glycoforms, etc. © 2004 Large Scale Biology Corporation. All Rights Reserved Worldwide

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Summary & Conclusions ƒ LSBC’s novel biomanufacturing platform can produce biologicals … … meeting stringent specification criteria both in novel and follow-on biologics programs … in commercially relevant scale … for competitive COGS … with capital economies relative to traditional production systems … with process FTO.

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