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 LSBC PROPRIETARY – 41006 - 1
Presentation outline
- Technology/platform background
- Applications
- Case study: Development of a follow-on product
FTO
CMC/product quality
- Lessons learned: Regulatory experience
Product-focused biopharmaceutical company
Strengths: Development Î Manufacturing
Areas: Therapeutic proteins and vaccines
Vacaville, California Owensboro, Kentucky

Headquarters; Product Development Process Development; cGMP Biomanufacturing
Large Scale Biology Corporation (LSBC)
GENEWARE® plant viral gene expression & biomanufacturing
platform
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)
Biomanufacturing of follow-on molecules
- r-Aprotinin
- r-Interferon alpha 2a and 2b
Industry Preferences – Biomanufacturing of FOBs
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
Potentially Blocking US Process Patents for r-INF-alpha
US Patent No. Assignee Comments Expiration
1 6,610,830 Genentech Microb. production of rhIFN 2020
2 6,482,613 Genentech Microb. production of rhIFN 2019
3 6,432,677 Genentech Microb./animal IFNs 2019
4 6,391,296 Toray IFN stabilization/E. coli 2019
5 6,225,455 PBL Biomed. Fusion prots./rhIFNs 2018
6 5,831,023 Genentech Porcine & bovine IFNs 2015
7 5,827,694 Genentech DNA & vectors for IFNs of
non-human origin/E. coli 2015
8 5,710,027 Boehringer Vector & process for
Ingelheim expr. IFNs in E. coli 2015
9 5,646,037 Ciba-Geigy Yeast vectors for rIFNs 2014
10 5,196,323 Boehringer rIFN made in E. coli; purific.
Ingelheim by affinity chromatography 2010
Potentially Blocking US Process Patents for rh-G-CSF
US Patent No. Assignee Comments Expiration
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
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)
GENEWARE® Tobacco Mosaic Virus (TMV) Soluble (Free) Protein
Viral Vector
Product Coat protein
Cap structure Replicase / transcriptase genes Movement gene gene
protein gene
RNA single plus (+) strand In plant hosts, the movement

viral genome 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 Wide variety of soluble (free)

the Tobacco Mosaic Virus (TMV) protein products produced
rod-shaped virion.
with high efficiency
Virus-Like Particle (VLP) TMV Coat Protein Fusion Viral Vector
Coat protein gene fused
to peptide at
Movement one of three locations
Cap structure Replicase / transcriptase genes
protein gene
RNA single plus (+) strand N-terminal fusion

In plant hosts, the movement
viral genome protein translocates virions
Loop region fusion
systemically to ensure high yield
Peptides, fused at C-terminal fusion

the gene level to
the coat protein
of TMV, are arrayed
for display on the viral
surface.
Over 2,100 peptides

RNA
can be displayed helix
on the surface of each
virion, forming hybrid
TMV-peptide Virus-Like
Particles (VLP) which
can be efficiently loop N terminus
manufactured and C terminus
purified. Schematic showing part of
the Tobacco Mosaic Virus (TMV) Schematic showing two adjacent coat proteins
rod-shaped virion. and N-, Loop- and C-terminal fusion options for
displaying peptides on the TMV virion surface.
LSBC’s Expression Vectors
Product
Replicase Movement
Cap structure (eg. GFP) Coat
protein
Normal Light
2 dpi 4 dpi 6 dpi
Product
(eg. GFP)
UV Light
Plant Cell Structure
Targeting of Polypeptide to Intracellular Compartments
Non-Specific and Specific

Accumulation of Protein
in Selected Plant Cell
Compartments
Enables Rational Product Extraction and Purification

Targeting of Polypeptides To The Apoplast
Downstream Processing Optimization
Plants secrete only Recovery of Protein
~1% of their total by Vacuum Infiltration
cellular protein into
the interstitial space Very little carryover
between cells of host proteins into
the API stream
Very simple purification

Apoplast targeting Dramatic reduction in COGS
enables product
concentration in a Proteins so recovered
fluid devoid of most
- Are correctly folded
host proteins
- Meet purity specs
- Have entered the clinic
Apoplast (interstitial space)
Features of Viral Expression
Speed Production cycle is 2-3 weeks duration (indoors)
- No plant breeding required
Copy Number Tens of thousands of copies of transcript per cell

- Chloroplast transformation: 10,000 cpy/cell
- Nuclear transformation: 1-2 cpy/cell
Purification Product can accumulate in plant cell

compartments, or on the vector itself
- More flexible extraction/purification options
Features of Viral Expression (continued)
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
Features of Viral Expression (continued)
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 vector-
derived contaminants
Indoor Manufacturing: Greenhouse Complex
Field-Scale Protein Manufacturing
Outdoor cultivation and viral

inoculation under USDA permits
Policy clearances to 1,000 acres
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
Biomass Homogenization Extract Clarification
Clarification
Chromatography/Ultrafiltration Finish and Fill
LSBC’s Product Development and Manufacturing
All Manufacturing Process Modules and Components in Place Today
Protein Expression
Quality and Regulatory Technologies Analytical Development
Quality Control Activity Assays
Quality Assurance Product Identity
Quality Systems Product Purity
FDA Submissions
Process Development
Fill & Finish
Bench-scale
Class 100 Facility
Pilot-scale
Sterile Vial Fill LSBC Biomanufacturing Manufacturing-scale
Product Purification
Clarification Agronomics
Filtration Field Crop Production
Chromatography Product Extraction
Greenhouse Production
Homogenization
Agriculture
Interstitial Fluid
USDA Regulatory
US Regulatory Agency Oversight
Summary: GENEWARE Environmental Releases
First Recombinant Virus Release in 1991
Over 15 Releases, 3 States, 198 Acres
Released Tested Over 17 Unique Products
First Field Release of Genomic Libraries
2004: Of 44 Acres Approved Nationwide, LSBC had 35 (80% )
2004: Used as Training Site for USDA Inspectors
No Adverse Public Reaction
Viral Expression of r-Aprotinin in Plant Hosts
Protease inhibitor Arg
Tyr Phe Tyr
Asn
6511 Da protein Ile

Ile Ala
Lys
58 amino acids Arg Ala
Val
3 disulfide bonds Ala
Gly
Tyr
Phe
Thr
Gly
Well characterized Lys Gly Phe

Lys
Gln Leu
Asn
Activity from Cys Cys
Ser
Cys
Asn
molecular Pro
Arg
Ala Asp
Cys
properties alone Gly

Ala
Lys
Arg Glu
Met
Market expansion Thr

Arg
Marketed product projected from Tyr Glu Leu

Thr
Bayer’s Trasylol® existing and new Pro Pro

Phe
Cys Cys
Bovine sourced indications, from Asp

Gly
current $180 M/yr Arg Pro

Ala
Gly
(ann.)
r-Aprotinin Extraction and Purification
Plant Propagation
Chromatography
Master Plasmid
Bank Separations
Inoculation
Working Plasmid Ultrafiltration and
Bank Diafiltration
Plant Harvest and Extraction

Transcript and
Virion Stocks 0.2 µM Filtration
Clarification
Host Seed
Bulk Fill
Ultrafiltration and
Diafiltration
Test Methods and Results of Aprotinin Comparisons
Assay Comparative Attribute r-Aprotinin Trasylol
Identity by Tryptic Conforms with bovine lung

Digest MALDI-TOF aprotinin predicted tryptic Conforms Conforms
fragments and fragment
mass mapping
derivatives (84% amino acid
coverage)
Identity by 6,512 Da ± 0.05% 6,512 Da 6,512 Da

MALDI-TOF MS
Identity by Amino Conforms with bovine lung Conforms Conforms

Acid Analysis aprotinin amino acid composition
Purity by Purity >99% >99%

SDS-PAGE
Circular Dichroism Identity/Structure Comparable Comparable
Purity by GC/MS Purity Comparable Comparable

Small molecular levels of target levels of target
weight host toxicants compounds compounds
Purity by Appearance Clear, colorless, free of visible Clear, colorless, Clear, colorless,
particles particle free particle free
Potency by Specific >6,500 KIU/mg protein 7,145 KIU 7,143 KIU

Activity
r-Aprotinin Biomanufacturing Summary
Things you can control well …
r-Aprotinin can be manufactured at scale (100s of Kg API per
year) using transient plant viral expression
The API conforms to the innovator product
The impurity profile of r-Aprotinin is “the same but better” than
the innovator product’s
LSBC’s “Animal-Free” r-Aprotinin is currently sold for non-
pharma applications (R&D, cell culture) through Sigma-Aldrich
Pre-IND Meeting with FDA
Things you can control less well …
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
Purpose of the Pre-IND Meeting
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)
The secondary purpose was to discuss an “abbreviated”
regulatory approval route such as 505(b)(2) or 505(j)
Purpose of the pre-IND Meeting
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
r-Aprotinin Development Strategy
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
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
Regulatory Issues and Guidance
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,
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
Immunogenicity Issues and Guidance
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
Indication and Label Issues
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
Comments Regarding “Follow-On Biologics”
There is currently no regulatory path to accommodate
“biogenerics”
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)
Comments Regarding “Follow-On Biologics”
The Agency is being encouraged to find “examples” of products
that may serve as models for “follow-on” development pathway
The Agency has openly commented regarding immunogenicity

and comparability issues around “follow-on biologics”
development
Aprotinin is a small protein of relatively low complexity
The FDA concurred with an abbreviated pathway partly based on

ability to manufacture and characterize this small protein
There are numerous advantages and reasons for moving away

from the bovine-sourced aprotinin
Molecular Complexity and Regulatory Complexity
Simpler, More Easily Characterizable Molecules Will Likely Move Faster
Through the Regulatory Process GM-CSF 127 / ~17,000 Da
EPO 165 / 30,400 Da
Molecule / No. aa / MW in Daltons
Interferon 166 / 22,500 Da
Manufactured by LSBC
G-CSF 175 / 18,800 Da
Bivalirudin 20 / 2,180 Da
scFv 240 / 27,000 Da
Glucagon 29 / 3,483 Da
Fab 450 / 52,000 Da
Nesiritide 32 / 3,464 Da
hGH 191 / 22,125 Da
Reteplase 355 / 39,571 Da
Enoxaparin ps / ~4,500 Da Imiglucerase 497 / 60,430 Da
Octreotide 8 / 1,019 Da Insulins 51-54 / 5,800
tPA 527 / 58,000 Da
– 6,100 Da
Eptifibatide 8 / 832 Da Lysosomal Acid Lipase 378 / 50 kDa
Aprotinin 58 / 6,512 Da
Leuprolide 9 / 1,269 Da α-Galactosidase 400 / 101 kDa
Lepirudin 65 / 6,979 Da
Desmopressin 9 / 1,183 Da MAbs >1,000 / 145-155 kDa
Increasing molecular complexity: # of amino acids, MW, folding, glycoforms, etc.

Summary & Conclusions
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.

Biologic Usa 10 2004

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Biologic Usa 10 2004

Enviado por

Direitos autorais:

Formatos disponíveis

Plant-Based Manufacturing of Biosimilar

and Next-Generation Biologics

Daniel Tusé, Ph.D.

Vacaville, California Owensboro, Kentucky

– API/impurity profiles may not be the same as innovator’s

US Patent No. Assignee Comments Expiration

1 6,521,449 GSF Microb. production of rh-G-CSF 2019

RNA single plus (+) strand In plant hosts, the movement

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

Product of interest is produced through subgenomic

Schematic showing part of Wide variety of soluble (free)

RNA single plus (+) strand N-terminal fusion

Peptides, fused at C-terminal fusion

Over 2,100 peptides

Non-Specific and Specific

Enables Rational Product Extraction and Purification

Very simple purification

 Copy Number Tens of thousands of copies of transcript per cell

 Purification Product can accumulate in plant cell

Viral vectors are not persistent in soil

Host plants used are Nicotiana species

APIs manufactured via viral vector technology are

Outdoor cultivation and viral

LSBC Biomanufacturing Campus, Owensboro, Kentucky, USA

Facility: 30,000 sq ft. Throughput: 3 metric tons of plant biomass/hr

 6511 Da protein Ile

 Well characterized Lys Gly Phe

properties alone Gly

 Market expansion Thr

 Marketed product projected from Tyr Glu Leu

Bayer’s Trasylol® existing and new Pro Pro

 Bovine sourced indications, from Asp

current $180 M/yr Arg Pro

Plant Harvest and Extraction

Identity by Tryptic Conforms with bovine lung

Identity by 6,512 Da ± 0.05% 6,512 Da 6,512 Da

Identity by Amino Conforms with bovine lung Conforms Conforms

Purity by Purity >99% >99%

Circular Dichroism Identity/Structure Comparable Comparable

Purity by GC/MS Purity Comparable Comparable

Potency by Specific >6,500 KIU/mg protein 7,145 KIU 7,143 KIU

 LSBC requested a pre-IND meeting with FDA to

 The primary purpose of the meeting was to discuss an

Interestingly, the Agency was not focused on the

 The Agency provided regulatory and clinical guidance, including

- The API manufacturing process is defined and reproducible

 With regards to the 505(b)(2) approval pathway for

NOTE: The primary obstacle to 505(b)(2) is the

 With regards to assessment of seroconversion and

 The Agency has openly commented regarding immunogenicity

 Aprotinin is a small protein of relatively low complexity

 The FDA concurred with an abbreviated pathway partly based on

 There are numerous advantages and reasons for moving away

Increasing molecular complexity: # of amino acids, MW, folding, glycoforms, etc.

… in commercially relevant scale

… for competitive COGS

… with capital economies relative to traditional production systems

… with process FTO.

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Copy Number Tens of thousands of copies of transcript per cell

Purification Product can accumulate in plant cell

6511 Da protein Ile

Well characterized Lys Gly Phe

Market expansion Thr

Marketed product projected from Tyr Glu Leu

Bovine sourced indications, from Asp

LSBC requested a pre-IND meeting with FDA to

The primary purpose of the meeting was to discuss an

The Agency provided regulatory and clinical guidance, including

With regards to the 505(b)(2) approval pathway for

With regards to assessment of seroconversion and

The Agency has openly commented regarding immunogenicity

Aprotinin is a small protein of relatively low complexity

The FDA concurred with an abbreviated pathway partly based on

There are numerous advantages and reasons for moving away