Download Biopharmaceuticals

Biopharmaceuticals
Protein Therapeutics
Andreas Castan
DISCLAIMER
In order to utilize the ‘Safe Harbor’ provisions of the United States Private Securities Litigation Reform Act of 1995,
Biovitrum is providing the following cautionary statement. This presentation contains forward-looking statements with
respect to the financial condition, results of operations and businesses of Biovitrum. By their nature, forward-looking
statements and forecasts involve risk and uncertainty because they relate to events and depend on circumstances that
will occur in the future. There are a number of factors that could cause actual results and developments to differ
materially from that expressed or implied by these forward-looking statements. These factors include, among other
things, the loss or expiration of patents, marketing exclusivity or trade marks; exchange rate fluctuations; the risk that
R&D will not yield new products that achieve commercial success; the impact of competition, price controls and price
reductions; taxation risks; the risk of substantial product liability claims; the impact of any failure by third parties to
supply materials or services; the risk of delay to new product launches; the difficulties of obtaining and maintaining
governmental approvals for products; the risk of failure to observe ongoing regulatory oversight; the risk that new
products do not perform as we expect; and the risk of environmental liabilities.
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Outline of the presentation
• Basics: Drug development and protein therapeutics
• Proteins vs. small molecules
• Protein drug discovery
• Protein drug development
– ReFacto show case
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Drug development in general
• Safety
– Acceptable therapeutic window
– No side effects
• Efficacy
– Effect in the chosen indication
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2
The Process
BLA
Market
Biotech
Pharma
Pharma
Biotech
C
O
S
T
Pharma
Biotech
H2L
Academia
Basic Research
Target ID
-hypothesis
Phase 3
Effect
Phase 2
Dose
Phase 1
PK/PD
Lead
Opt
Candidate
Candidate
Drug
Drug
Screening
Validation
Discovery
Pre-Clinical
Clinical
RISK
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Protein Therapeutics
• Proteins are fragile i.e. cannot be autoclaved
• Normally injected intravenous or subcutaneous
– A number o new devices under development
• Produced by
– Bacteria or mammalian cells
– Plasma or other biological sources
– Transgenic animals or plants
• Replacement therapy or new, engineered proteins
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3
Outline of the presentation
• Basics: Drug development and protein therapeutics
• Proteins vs small molecules
• Protein drug discovery
• Protein drug development
– ReFacto show case
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NBEs versus NCEs inherent differences, but good complementarity's!
Therapeutic proteins (NBEs)
+•
-
protein-protein interactions
Small molecules (NCEs)
+•
•
secreted natural proteins - high eff./pot./sel, less sideeffects
•
engineered non-natural proteins
•
multiple functions via fusion proteins
•
injectables
•
non-natural proteins - immunogenicity
•
extra cellular & mainly peripheral targets
oral administration
• central & peripheral - intra
& extra cellular targets
• protein-protein interactions
-
• toxicity, selectivty, side effects
• some “mimetics” hard to develop...
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4
NBEs versus NCEs differences in development times!
¾ Historically NBEs show
• Shorter development times
• Replacement therapy
• Defined pharmacological action
• More successful in clinical development
• Orphan drug approval
• Treatment of rare, severe diseases
• Easier to prove efficacy
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NBEs versus NCEs convergent trend in development time!
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12
10
8
6
4
NBEs (n=47)
2
NCEs (n=511)
19
99
19
97
19
95
19
93
19
91
19
89
19
87
0
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85
Mean development time [years]
Mean development times
Year of launch
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5
NBEs versus NCEs convergent trend in development time!
Why ?
¾ Nature of NBEs affects the development time
• Replacement
therapy
• Engineered proteins
with altered functions
• mAbs and antibody fragments
• Vaccines
¾ Little difference in FDA mean approval time
¾ NBEs still more successful in Ph I-II but failure increasing
in Ph III
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Outline of the presentation
• Basics: Drug development and protein therapheutics
• Proteins vs small molecules
• Protein drug discovery
• Protein drug development
– ReFacto show case
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6
The NBE Discovery Process involves
Target
ID
Lead selection
Lead
Generation
CD
Lead
Preclinical Phase I Phase II Phase III
Optimization
Protein Activity
Relationship (PAR)
Primary
sequence
• Protein Optimization
• Protein Design
• Quantification assay - Protein Engineering
• Protein Production - Fusion partner
for preclinical testing • Pharmacokinetic
• Screening funnel – analysis
- Stability / half-life
in vitro and in vivo
Reg
Posttranslational
modifications
GMP process
• Optimization/characterization of
- PEGylation
- Glycosylation
• Process development – PI/II studies
• GLP-toxicity, immunogenicity
• Formulation
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Outline of the talk
• Basics: Drug development and protein therapeutics
• Proteins vs small molecules
• Protein drug discovery
• Protein drug development
– General development needs
– Monoclonal Antibodies
– ReFacto show case
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7
Documentation to support approval of a new
drug
• Description of molecule
• Mechanism of action
• Manufacturing and control
– Drug Substance (Active Pharmaceutical Ingredient)
– Drug Product
• Preclinical data (animal studies)
• Clinical data
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Two stage Process Development
Pre clinical
Tox batch
Phase I batch
Phase II batch
Phase III batch
Manufacturing
Minimal process development
Focus on few important parameters
Focus on in-process controls and analytical methods
Focus on impurity profile and impurity characterization
Extensive process development
Optimization and justification of all parameters and
extensive work on critical parameters
Focus on yield, stability, reproducibility and scalability
Reducing number of in-process control and spec. analyses
Phase I/II process
Intermediate yield
Intermediate purity
Limitied # of batches
Manufacturing process
High yield
Low cost
High purity
Robust
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Additional information required in drug
development projects
• Market needs (sets scale of manufacturing)
• Competitor situation
• Intellectual Property (IP, patents)
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Currently used techniques for Recombinant
Biopharmaceuticals
• Design of molecules by genetic engineering
• Expression in bacterial and mammalian hosts
– E.coli
– Pichia pastoris
– CHO & NS0
• Development of cell lines with high expression levels
• Host modification / cell line optimization
– Removal of proteases and impurities
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Fed batch cultivation of mammalian cells
Glucose + Glutamine + Feed medium
50L
25L
5L
150L
10-11 days
4 days
16 days
Cell thaw and expansion
Seed train
Production
400L
Purification
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Over 300 monoclonal antibodies under
development
• Design of new molecules
• Design of new monoclonal antibodies
• Design of antibody fragments
• Expression of monoclonal antibodies
• Mammalian production 0,5-4g/L
• Production scale up to 20 000 L Bioreactors
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MAb heterogeneity
Identity
N-terminal
C-terminal
Modifications
-Deamidation
-Oxidation
-Fragmentation
Binding
Aggregation
Sulfhydryl groups
Disulphide bridges
Glycosylation
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Product Flow
Cell Bank
Cultivation
Filling/Freeze drying
Distribution
Purification
Labeling
Packaging
Marketing
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New era in Biotechnology?
• 5 out of 20 top products are biologics
• Price for yearly treatment with Refacto 75 KUSD
• Needs to be decreased to some hundreds/ thousand USD/year
• Platform approach upstream and downstream
• Demand vs capacity ongoing discussions
• Robust processes
• Demand for high titers 5 g/L or even 10
g/L
7000
6000
total demand biologics (kg/year)
• The biotech industry today: A shift towards
antibodies
5000
4000
3000
2000
1000
0
2002
2003
2004
2005
2006
year
Source: UBS investment research
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Upstream productivity increase: Focus on DSP
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12
Eliminate (DSP) bottlenecks!
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Challenges for modern downstream process
• Technical challenge
– Complex and technically demanding with inherent complexity, high initial volume and low product titer
– Need for robust and scalable process for the entire DSP
– Increasing regulatory focus on DSP and comparability
• Financial challenge
– Processes are fixed cost driven (investment vs consumables)
– Manufacturing cost 15-25 % of total COGs
– Costs for DSP up to 75 % of manufacturing costs
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Major process derived contaminants in Mab production
FDA PTC 1997
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Design of candidate drug
• Interacting with target
• Known mechanism
• Safety
• Tolerability
• Efficacy
• Immunogenicity
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ReFacto (Recombinant Factor VIII SQ)
Gene Construction
90 kDa
H2N
A1
1
110 kDa
A2
80 kDa
B
A3
740
SQ-link
1649
C1 C2
COOH
2332
SFSQNPPVLKRHQR
741
745
1637
1648
In vivo processing
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ReFacto (Recombinant Factor VIII SQ)
Processing of Primary Translation Product
170 kDa
A1
A2
SQ-link
A3
C1 C2
SFSQNPPVLKRHQR
Intracellular Processing
90 kDa
A1
80 kDa
A2
A3
C1 C2
Me 2+
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Structure of Factor VIII and r-VIII SQ
Factor VIII
full-length
r-VIII SQ
B-domain deleted
Heavy Chain
Heavy Chain
A2
A3
A3
A1
Light
Chain
C2
2+
Me
Me2+
C1
Light
Chain
B
C1
Me2+
A2
A1
C2
PL-binding site
vWF-binding site
PL-binding site
vWF-binding site
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Development of manufacturing system
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ReFacto Manufacturing Process
CHO Cultivation
Continuous Harvesting
Primary Capture
Ion exchange
Downstream Purification
Drug Substance
Viral inactivatin
Immunoaffinity
Formulation
Ion exchange
Lyophilization
HIC
Buffer exchange
Drug Product
Packaging
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ReFacto is produced with
recombinant gene technology
Chinese Hamster Ovary, CHO, cells
is used as host cell
The human gene for
Factor VIII has been
incorporated in the CHO
cell DNA, and the cell
has been cloned
Factor VIII gene
DNA
The CHO cells
produce
human Factor VIII,
ReFacto
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Cell bank preparation
Clone
Master Cell Bank
MCB
Cultivation Process
Working Cell Bank
WCB
Post Production
Cell Bank, PPCB
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ReFacto Cell Culture: Production medium
500 L
medium / day
Well defined
60 components
Nutritionally rich
Antibiotics free
Serum free
(Albumin Ph. Eur.)
Recombinant insulin
500 L spent
medium / day
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ReFacto Cell Culture: Process Flow
Cell bank
(WCB)
T-Flask
Spinner
50L
500L
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ReFacto Purification Process
Harvest filtrate
Ion Exchange Chromatography
Virus Inactivation
Immuno Affinity Chromatography
Ion Exchange Chromatography
Hydrophobic Interaction Chromatography
Gel Permeation Chromatography
Drug Substance
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Manufacturing quality
• Identify critical parameters
– robustness studies (process characterization)
• Batch records and Standard Operating Procedures (SOPs)
• Cleaning validation
• Preventive maintenance programs
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ReFacto, Characterization
• Identity
• Purity
• Structure
• Function
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ReFacto
Purity tests
• Specific activity
• CHO cell protein
• SDS-PAGE
• Mouse IgG
• SEC-HPLC
• TNBP
• RP-HPLC
• Triton X-100
• DNA
• Ethylene glycol
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ReFacto
Analysis of structure
• Amino acid composition
• SDS-PAGE/ Western Blot
• Amino acid sequence
• Tryptic map
• Carbohydrate composition
• Gel filtration
• Carbohydrate sequence
• MALDI-MS
• RP-HPLC
• Circular dichroism
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ReFacto
Analysis of structure
• Amino acid composition
• SDS-PAGE/ Western Blot
• Amino acid sequence
• Tryptic map
• Carbohydrate composition
• Gel filtration
• Carbohydrate sequence
• MALDI-MS
• RP-HPLC
• Circular dichroism
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Analytical package
• Identify assays
• Develop specifications
– drug substance and Finish Product
• Validation of assays
• Transfer to Quality Control, QC, laboratory
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ReFacto
Pharmaceutical Formulation
• Stable freeze-dried formulation
• No protein stabilizer
• Small reconstitution volume
• Short dissolution time
• High recovery
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Recovery during formulation and freeze-drying
300
Before filtration
After filtration
Thawed 5 times
Freeze-dried
VIII:C (IU/ml)
250
200
150
100
50
0
Buffer only
Polysorbate 80
PEG 4000
HSA
Haemaccel
Haes
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ReFacto Storage Stability
• Lyophilized product
– 36 months 2 to 8oC
– whereof 6 months at room temperature <25oC
• Reconstituted product
– 3 hours at <25oC
– stable for 48 h in syringe system for continuous infusion
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Virus Safety
• Cell bank testing
• In-process testing
• Validation of purification process
– model virus studies
Virus reduction by filtration:
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ReFacto virus validation data
Log reduction
Process step
Enveloped
MXR
yes
MCF
yes
IBR
yes
PI3
yes
SD treatment
≥ 4,6
≥ 4,1
≥ 4,9
≥ 6,2
5,1
5,5
≥ 7,9
6,3
Immunoaffinity
chromatography
Gel filtration
Overall reduction
POL
no
3,8
1,8
≥ 9,7
≥ 9,6
≥ 12,8
≥ 12,5
5,6
MXR, murine xenotropic retrovirus (Retroviridae)
MCF, mink cell focus virus (Retroviridae)
IBR, infectious bovine rhinotracheitis virus (Herpesviridae)
PI3, parainfluenza 3 virus (Paramyxoviridae)
POL, polio virus (Picornoviridae)
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Safety and efficacy
• Preclinical studies
– Rat (Sprague-Dawley)
– Hemophilia A dog
– Monkey (Cynomolgus)
• Clinical trials
– PTP, Previously Treated Patients
– PUP, Previously Untreated Patients
– Surgery
– On-demand/prophylaxis
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Regulatory process
• IND, Investigational New Drug
• BLA, Biologics License Application
– FDA, US
• MAA, Market Authorization Application
– Centralized procedure, EMEA, EU
• Rest of the World Applications
• Comparability protocols & variations
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Commercial phase
• Ensure manufacturing capacity
• Life cycle management programs
– Convenient devices
• Current GMP
• Market support
• Regulatory support
• Phase IV clinical studies
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Abbreviations
• BLA – Biologics License Application
• MAA, Market Authorization Application
• CD – Candidate drug
• MCB - Master Cell Bank
• CTD – Common Technical Document
• NBE – new biological entity
• EMEA- European Medicinal Evaluation
Agency
• NCE – new chemical entity
• FDA- Food and Drug Administration
• PD - Pharmacodynamics
• GLP - Good Labboratory Practice
• PK- Pharmacokinetics
• GMP – Good Manufacturing Practice
• PPCB – Post Production Cell Bank
• H2L- Hit to lead
• SOP – Standard Operation Procedure
• IND – Investigational new drug
• WCB - Working Cell Bank
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Andreas Castan Biovitrum AB
E-mail: [email protected]
www.biovitrum.com
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www.biovitrum.com
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