Estell

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.