Adapting Industry Practice for Rapid Large Scale Manufacture of Pharmaceutical Proteins
David A. Estell, Ph.D.
Genencor International
Rapid Large Scale Manufacture of Pharmaceutical Proteins
• Current manufacturing processes for pharmaceutical proteins are low volume, high cost and slow to start.
• High volume ( up to 100 million doses) production of a novel protein drug within weeks requires a radical change to these processes.
Genencor at a Glance
• History traced to 1982 - joint venture of Genentech and Corning
• $390 Million in total revenues during 2004
• Among the world’s largest biotech companies
• 8 manufacturing sites; ~4 million liters of capacity
Pharmaceutical Protein Production
• Selling price > $1000/gram active protein
• Volumes < 10,000 gram active protein /month
• Full scale manufacturing more than 1 year after creation of final molecule
Genencor: Industrial Protein Production
• Selling price < $1/gram active protein
• Volumes > 30,000,000 gram active protein /month
• Full scale manufacturing within weeks of creation of final molecule
Rapid, Large Scale Pharmaceutical Protein Production
The tools and methods of industrial biotechnology can be used to produce several million doses of a protein pharmaceutical within weeks of identification.
Industrial costs and volumes enable topical, oral or inhaled delivery systems.
THE TIME IS NOW.
Genencor Approach to Protein Production
• Identify protein scaffold• Choose gene/host system • Develop high yield fermentation process• Design robust, rapid and efficient
recovery process• Create formulation and delivery system• Engineer the scaffold to provide the
desired properties
Protein Scaffolds
• Multi-Domain Fusion Proteins
• Monoclonal Antibody
• Viral or Bacterial Coat Protein
• Inhibitor
• Enzyme
Catalytic region
Binding Domain
Native Enzyme
Linker
Fusion Protein Scaffolds
Native Enzyme is a multi-domain protein secreted at very high levels. The Native enzyme directs the fusion protein through the secretion machinery to give properly folded, secreted product protein
Catalytic region
Fusion Protein
Linker
Fusion Protein Scaffolds
The desired protein is secreted at high levels into clean fermentation media. The protein is properlyfolded and may be processed from the fusion protein in the fermentor or at a later step.
Processing site
Desiredprotein
Multitude of Systems for large scale Efficient Expression of Proteins:
Bacterial Examples:• Escherichia coli• Bacillus subtilis• Bacillus licheniformis
Fungal Examples: • Aspergillus niger• Trichoderma reesei
Efficient Host Construction Rapid Fermentation Process Development Efficient Downstream Processing
Commercially Viable Formulations Ease of Scale / Tech Transfer Robust Competitive Cost Position
Gene/Host System
Advantages of Microbial Systems
• Speed to construct stable production strains.• Ability to screen for improvements in same
host used for manufacturing.• No animal products required during
production.• Short fermentation time, robust process up to
very large scale.• Reduced capital expenditure. Reduced cost
of goods sold.
Frozen
Seed
Seed Fermemt
er
Production Fermenter
Seed Flas
k
Pre-Seed Ferment
er
Typical Fermentation TrainTotal time 3-20 days
Fermentation Process
Protein Recovery Processes
• Filtration• Extraction• Large Scale
Chromatography• Crystallization
Glucose Isomerase Crystals
Glucose Isomerase Crystals
High-Throughput Process Development for Purification of Recombinant Proteins
• High-throughput microtiter plate recovery process development
• Scalable screening technique • Appropriate analytical methods to enable rapid
analysis of screening results
Create formulation and delivery system
Protein stable in formulation for months at > 40°C.Formulations may be solid or liquid.Release of product may be controlled.Formulations are food grade.
Typical Microbial Production Process
• Create Production Host– 2-4 weeks
• Fermentation Process– 3-20 days
• Recovery Process/Formulation– 2-10 days
Rapid Protein Drug Production
• Expression system can be in place for each protein scaffold.
• Each scaffold can be engineered to have the basic properties required.
• Immunogenicity• Stability• Pharmakokinetics
• A high yield fermentation and recovery process can be put in place for each scaffold.
• Formulation and delivery system in place for scaffold protein.
Rapid Protein Drug Production
For Protein drugs a small number of sequence changes in a protein scaffold will give the desired properties:– Binding site in an antibody– Epitopes in a viral or bacterial coat
protein– Enzyme/Receptor binding site in an
inhibitor
Rapid Protein Drug Production
• Protein drug identified (e.g. by sequence of pathogen)
• Protein drug created through engineering one of the already developed protein scaffolds
• New protein drug is produced using processes put in place for the protein scaffold
Rapid Protein Drug Production
• Proteins for which gycosylation is not required for activity can be produced now.
• Proteins for which glycosylation is key may require additional host engineering or post production modification
Rapid Protein Drug Production
Products with individual dose sizes of < 100 mg (e.g vaccines) can be made with existing technology and capacity.
Products with dose sizes > 100mg (e.g. monoclonal antibodies) may require initial yield improvement for the scaffold protein
Fermentation capacity (8 weeks) for 100,000,000 doses @ 1mg/dose
Yield (g/L)1 100,000 L
142,857 L @ 70% recoveryfermenter volume working volume (L) L/8wks/ferm # of fermenters L/8wks/ferm # of fermenters
3 day turnaround 10 day turnaround3,000 2,400 45,600 3 14,400 10
30,000 24,000 456,000 1 144,000 1360,000 288,000 5,472,000 1 1,728,000 1
10 10,000 L14,286 L @ 70% recovery
fermenter volume working volume (L) L/8wks/ferm # of fermenters L/8wks/ferm # of fermenters3 day turnaround 10 day turnaround
3,000 2,400 45,600 1 14,400 130,000 24,000 456,000 1 144,000 1
360,000 288,000 5,472,000 1 1,728,000 1
Rapid Protein Drug Production
• Current production processes are capable of producing 100,000,000 g of protein in < 12 weeks.
• Yield drives fermentation capacity• Yield needs to be at least 1 g/L to meet
the timelines• Fermentation and recovery processes
need to be in place
Rapid Protein Drug Production
Time and volume targets can best be achieved by developing robust processes for expression (> 1g/L), fermentation and recovery of the scaffold protein. These processes would then be used for the engineered final product.
Rapid Protein Drug Production
Example: fungal production of a monoclonal antibody
Filamentous Fungi for Pharmaceutical Production
• More than $ 13 billion/year of injectable and oral antibiotics are produced through fungal fermentation.
• The published yields are 1-50 g/L
• Recovery processes can be used for proteins
• Sales prices are $1-$100/g
GA-heavy chain(1) 50 kDaKR
GA-light chain ()
25 kDaKR
55 kDa
Strategy for Ab Production in Aspergillus
Glucoamylase (GA) Catalyticdomain
SBDLinker
Glucoamylase (GA) Catalyticdomain
SBDLinker
55 kDa
GA-Ab fusion
IgG1k
Assembly and Processing of Ab in Aspergillus at 1g/L
Appl Environ Microbiol. 2004 May;70(5):2567-76.
• Hydrophobic Charge Induction chromatography will capture Ab and separate it from glucoamylase-Ab fusion in a single step.
S
N
4-Mercapto-Ethyl-Pyridine(4-MEP, pKa=4.8) - - -
MEP HYPERCELR
HCIC developed and patented by Genencor and Massey University.Commercialized for antibody purification by BioSepra and available from Ciphergen.
Elution from HCIC pH 4.5 = Free Antibody (B)pH <4 = Glucoamylase-Ab
fusion (A)
200
11697
6655
3631
21
kDa A B
Reducing SDS-PAGE
200
11697
6655
3631
21
kDa A B
Reducing SDS-PAGE
1st Step Purification by HCIC
2nd Step of Purification by SEC
EC50
15.4917.8218.18
Control Hu1D10Aspergillus Ab 1Aspergillus Ab 2
EC50
15.4917.8218.18
• There was no significant difference in affinity between NS0-derived and Aspergillus-derived antibody.
(250ng FITC-Control Ab)
Data from PDL, Inc.
Competition Binding Assay
1
10
100
0 5 10 15
H1H2H3H4
CHO Ab Fungal Abfh1fh2fh3
time days
Mean parameters from individual animals
CHO Ab
Fungal Ab
Parameter Units n=4 n=3
Cmax ug/mL 49.5(6.2) 48.3(3)
No_points_Lambda_z 8.25(1.5) 4.67(1.5)
AUC_obs day*ug/mL 153(4.4) 143(11)
HL_Lambda_z day 11.1(3.3) 15(2)
AUCINF_obs day*ug/mL 297(45) 285(5.4)
AUC_%Extrap_obs % 47.7(6.9) 49.8(3)
Vz_obs mL/kg 106(16) 152(23)
CHO and Fungal Ab Display Similar Pharmacokinetics in Rat
Rapid, Large Scale Pharmaceutical Protein Production
The tools and methods of industrial biotechnology can be used to produce several million doses of a protein pharmaceutical within weeks of identification.
Industrial costs and volumes enable topical, oral or inhaled delivery systems.
THE TIME IS NOW.