1. Adapting Industry Practice for
Rapid Large Scale Manufacture
of Pharmaceutical Proteins
David A. Estell, Ph.D.
Genencor International

2. 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.

3. 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

4. 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

5. 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

6. 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.

7. 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

8. Protein Scaffolds
• Multi-Domain Fusion Proteins
• Monoclonal Antibody
• Viral or Bacterial Coat Protein
• Inhibitor
• Enzyme

9. 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

10. Catalytic
region
Fusion Protein
Linker
Fusion Protein Scaffolds
The desired protein is secreted at high levels into
clean fermentation media. The protein is properly
folded and may be processed from the fusion protein
in the fermentor or at a later step.
Processing site
Desired
protein

11. 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

12. 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.

13. Frozen
Seed
Seed
Fermemter
Production
Fermenter
Seed
Flask
Pre-Seed
Fermenter
Typical Fermentation Train
Total time 3-20 days
Fermentation Process

14. Protein Recovery Processes
• Filtration
• Extraction
• Large Scale
Chromatography
• Crystallization
Glucose Isomerase Crystals
Glucose Isomerase Crystals

15. 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

16. 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.

17. Typical Microbial Production
Process
• Create Production Host
– 2-4 weeks
• Fermentation Process
– 3-20 days
• Recovery Process/Formulation
– 2-10 days

18. 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.

19. 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

20. 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

21. 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

22. 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

23. Fermentation capacity (8 weeks) for
100,000,000 doses @ 1mg/dose
Yield (g/L)
1 100,000 L
142,857 L @ 70%recovery
fermenter volume working volume (L) L/8wks/ferm # of fermenters L/8wks/ferm # of fermenters
3 day turnaround 10 day turnaround
3,000 2,400 45,600 3 14,400 10
30,000 24,000 456,000 1 144,000 1
360,000 288,000 5,472,000 1 1,728,000 1
10 10,000 L
14,286 L @ 70%recovery
fermenter volume working volume (L) L/8wks/ferm # of fermenters L/8wks/ferm # of fermenters
3 day turnaround 10 day turnaround
3,000 2,400 45,600 1 14,400 1
30,000 24,000 456,000 1 144,000 1
360,000 288,000 5,472,000 1 1,728,000 1

24. 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

25. 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.

26. Rapid Protein Drug Production
Example: fungal production of a
monoclonal antibody

27. 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

28. GA-heavy chain
(γ1) 50 kDaKR
GA-light chain (κ)
25 kDaKR
55 kDa
Strategy for Ab Production in
Aspergillus
Glucoamylase (GA)
Catalytic
domain
SBD
Linker
Glucoamylase (GA)
Catalytic
domain
SBD
Linker
55 kDa

29. GA-Ab fusion
IgG1k
Assembly and Processing of Ab
in Aspergillus at 1g/L
Appl Environ Microbiol. 2004 May;70(5):2567-76.

30. • 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 HYPERCEL
R
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
116
97
66
55
36
31
21
kDa A B
Reducing SDS-PAGE
200
116
97
66
55
36
31
21
kDa A B
Reducing SDS-PAGE
1st Step Purification by HCIC

31. 2nd Step of Purification by SEC

32. EC50
15.49
17.82
18.18
Control Hu1D10
Aspergillus Ab 1
Aspergillus Ab 2
EC50
15.49
17.82
18.18
• 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

33. 1
10
100
0 5 10 15
H1
H2
H3
H4
CHO Ab Fungal Ab
fh1
fh2
fh3
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

34. 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.